T.C.

TI

Esma YILDIRIM

Temmuz 2019

T.C.

TI

( )

H

Esma YILDIRIM

Birimi

TYL-2018-8300 kod ile

Temmuz 2019

ii

LUK

irtirim.

- : Uzm. Ecz. Esma YILDIRIM

v

Tezimin

ilerde de an sevgili

ve Dr. a,

Image J analizindeki

D e, Erciy

birimine, , hissettirdikleri sevgi,

destek , annem Hatice

YILDIRIM

Esma YILDIRM

Kayseri, Temmuz 2019

vi

Esma YILDIRIM

Temmuz 2019

:

M KH) osteoblastlara, adipositlere ve kondrositlere

V

inhibi sodyum

kanal inhibisyo

kappaB (NF- -

B'nin

inposetinin

vii

gr

MKH'lere

Alizarin Red S ile kalsifikasyonu Alkalen

Fosfataz (ALP) tayini i .

laveten RUNX2, ALP ve COL1A1 mRNA gen ekspresyon

- Vinposetin uygulanan gruplarda

RUNX2, ALP ve COL1A1 mRNA gen ekspresyon

zken,

azal . Bu azalma Alizarin Red S ve ALP boyama verileri

Bu

zellikle a vinposetin

etmek gerekebilir.

Anahtar Kelimeler:

viii

THE EFFECT OF VINPOCETINE ON OSTEOGENIC DIFFERENTIATION OF

MESENCHYMAL STEM CELLS

Esma YILDIRIM

Erciyes University, Health Sciences Institute,

School of Medicine,

Department of Medical Pharmacology,

Master Thesis, July 2019

Supervisor: Asst. Prof. Dr.

ABSTRACT

Mesenchymal stem cells (MSCs) can differentiate into osteoblasts, chondrocytes and

adipocytes, whereas hematopoietic stem cells into osteoblasts. Disruption of this

balance within the body has been associated with several diseases including

osteoporosis. Vinpocetine is used in many countries in the treatment of cerebrovascular

diseases, like stroke and dementia. Today, vinpocetine is also used as a dietary

supplement to improve memory. Vinpocetine inhibits phosphodiesterase 1 and voltage-

dependent sodium channels. In addition, it is known to inhibit nuclear factor kappaB

(NF- B) by the inhibition of se (IKK). In a study, it was shown that activation

of NF- B stimulates osteogenic differentiation of MSCs. In another study it was shown

that vinpocetine suppressed osteogenic differentiation of vasculer smooth muscle cells.

Based on these two studies, we aimed to investigate the effect of vinpocetine on

osteogenic differentiation of rat bone marrow-derived MSCs in vitro. We studied with

the clinical plasma concentration of vinposetine; , and also ,

which do not cause cytotoxic and apoptic effects on MSCs. Effect of vinpocetine was

observed on 5 groups as follows; control, differentiation medium group, and

vinpocetine treated groups. At the end of the 14 and 21 days, we analyzed by

immunohistochemical Alizarin Red S and Alkaline Phosphatase (ALP) stainings. In

addition, RUNX2, ALP and COL1A1 mRNA gene expression levels were measured by

qRT-PCR method. No significant difference was observed in the RUNX2, ALP and

COL1A1 mRNA gene expression levels in the groups treated with vinpocetine

ix

compared to differentation medium applied group on Day 14, however, RUNX2 and

ALP gene expression leve

vinpocetine treated groups. Staining results were in accordance with the gene expression

results. Vinpocetine has been shown to reduce osteogenic differentation of MSCs at

concentrations that do not affect cell viability

In conclusion elderly patients may require attention in terms of osteoporosis during the

treatment with vinpocetine, however, we need in vivo studies for to be sure the effect of

vinpocetine on osteoblastic activities.

Keywords: Vinpocetine; mesenchymal stem cells; osteogenic differentiation; cell

culture.

x

........................................................................................................................ i

................................................................................. ii

........................................................................................ iii

.................................................................................................................... v

............................................................................................................................... vi

ABSTRACT .................................................................................................................. viii

............................................................................................................... x

KISALTMALAR ............................................................................ xiii

T ................................................................................................. xvi

.................................................................................................. xvii

1. ....................................................................................................... 1

LER ................................................................................................... 2

.............................................................................................. 2

2.2. lerin ..................................................................... 2

lerin .................................................................... 3

............................................................... 4

2.3.1.1. Embriyo ............................................................ 4

............................................ 4

2.3.1.2.1. H .......................................... 4

............................ 5

......... 7

............................................... 7

............................................................... 7

xi

............................................................. 7

............................................................ 7

2.4. Vinposetin .......................................................................................................... 8

2.4.1 ..................................................................... 8

2.4.2. Vinposetinin ............................................................... 9

.......................................... 9

+ .................................... 10

..................................................... 10

2.4.3. Vinposetinin Etkileri ................................................................................. 12

ar .................................................................. 12

................................................................... 13

2 ......................................................................... 13

2.5. Osteoporoz ....................................................................................................... 13

......................................................................... 14

3. ............................................................................................. 16

Malzemeler ................................................. 16

3.2. c ............................................................................ 18

3.2.1. .................................................................................... 18

3.2.2. , ......................... 19

3.2.3. Karakterizasyonu .................................................................... 19

........................................................................ 19

istokimyasal Boyama .................................................. 20

3.2.4. a ; MTT Profilerasyon Testi .................................. 21

3.2.5. Annexin V/7-ADD ile Apoptoz Testi ........................................................ 21

3.2.6. Betagalaktozidaz Boyama Kiti ile Senesens Tayini .................................. 22

3.2.7. ................................................................. 23

3.2.7.1. Alizarin Red S Boyama .............................................................. 24

xii

3.2.7.2. ALP Boyama .............................................................................. 24

3.2.7.3. Oil O Red Boyama ..................................................................... 24

3.2.7.4. Image J P Analiz ...................................................... 25

3.2.8. ..................................................................... 26

......................................................................... 26

3.2.8.2. cDNA Sentezi ............................................................................. 28

3.3. Analiz ............................................................................................... 29

4. BULGULAR ............................................................................................................. 30

4.1. Karakterizasyonu ....................................................................... 30

4.2. MTT Testi ........................................................................................................ 32

4.3. Apoptoz Testi Verileri .................................................................................... 35

4.4. Senesens Analizi .............................................................................................. 36

4.5. ................................................................ 37

4.6. Gen Ekspresyon ......................................................................... 41

......................................................................................... 46

6. KAYNAKLAR .......................................................................................................... 51

EKLER ................................................................................................................... 64

EK 1. ........................................................................................................................ 64

................................................................................................................... 65

xiii

KISALTMALAR VE SIMGELER

ALP : Alkelen fosfataz

ATP : Adenozin trifosfat

AUC : Plazma konsantrasyonu

BCIP : 5-Bromo-4-kloro-3-Indolil Fosfat

BMP : Kemik morfogenetik proteini

cAMP : Siklik adenozin monofosfat

cDNA : Komplementer DNA

CD73 : Cluster of Differentiation 73

CD90 : Cluster of Differentiation 90

CD34 : Cluster of Differentiation 34

CD45 : Cluster of Differentiation 45

CD79 : Cluster of Differentiation 79

CD105 : Cluster of Differentiation 105

cGMP : Siklik guanozin monofosfat

Cmax : Maksimum plazma konsantrasyonu

COL1A1 : Kollejen tip 1 alfa 1

CO2 : Karbondioksit

Ct :

DMSO

xiv

DNA : Deoksiribo n asit

DPBS : Dulbecco fosfat tamponlu salin

EDTA : Etilendiamin tetraasetik asit

EKH : Embriyoni

FBS

FITC : Floresein izotiyosiyanat

hADSC

HLA-DR -DR

HKH

IC50 yon

ISCT : Ulu

IKK : Protein kinaz kompleksi

IL

ISCT

MKH

ml : Mililitre

MTT : Metiltiazol difenil tetrazolyum

Na : Sodyum

NBT : Nitro blue tetrazolium

NF- B

OPG : Osteoprotegerin

xv

PBS : Fosfat tamponlu salin

PDE : Fosfadiesteraz

PE : Fikoeritrin

PPAR- - gama

RANK

RANKL

RNA : Ribo asit

RT-PCR : a ncir Reaksyonu

RUNX2 :

S.H. : Standart hata

SPSS

TNF- : is alfa

TNFR

: Mikrolitre

m : Mikrometre

: Mikromolar

7-AAD : 7-Aminoaktinomisin D

-AMP -Adenozin monofosfat

-GMP : 5 -Guanozin monofosfa

xvi

TABLOLAR L STES

50 .......................................... 11

................................................................................ 16

............................................................................... 17

isinde kullan ....................................... 20

.............................................. 23

.................................................................... 26

Tablo 7. MTT deneyi 24. 48. ve 72. saat verileri . .......................................................... 33

Tablo 8. 14. ve ........................... 41

Tablo 9. RUNX2, ALP, COL1A1 nisbi

mRNA gen ekspresyon .............................................................................................. 43

Tablo 10. RUNX2, ALP, COL1A1

nisbi mRNA gen ekspresyon ..................................................................................... 45

xvii

.................................................................................... 3

............................................ 6

................................. 6

ve vinposetinin kimyasal .......................................................... 8

............................................................ 12

analizleri .......................................................................................................................... 25

................................................................... 27

............ 31

..................................................................................................... 32

lizarin red S ve ALP

............................................................................. 32

MTT deneyi (24. 48. ve 72. saat) verileri. ....................................................... 35

Vinposetinin MKH'lere olan apoptotik etkisi .................................................. 36

................................. 36

Al ................................ 37

................................................ 38

MKH'lerin 14. ve 21. g

.................................................................................................... 39

ait invert mikroskop

...................................................................................................................... 39

................................................................................. 40

fa

nisbi mRNA gen ekspresyon grafikleri ........................................................................... 42

nisbi mRNA gen ekspresyon grafikleri ........................................................................... 44

1

1.

MKH) osteojenik

olarak bilinmektedir ve MKH'lerdeki

ektedir (Hu ve ark., 2018). Vinkamin alkaloidinin

vinposetin

antioksidan, vazodila etkili r (Zhang ve

ark., 2018). ndan kognitif fonksiyonu mak

beslenme takviyesi olarak .,

2000). Vinposetinin fosfodiesteraz 1A,1B,1C (PDE1A,1B,1C)

yonu ve I B kinaz (IKK) inhibisyonu

yoluyla NF- B) dan

(Zhang ve Yang, 2015).

v os

(Wang ve ark., 2016). - B

aktivasyonunun insan adipoz doku MKH'lerin osteojenik farkl

(Cho ve ark., 2010). Bu ya

kullan vinposetinin

far etkilerini

2

2. LER

2.1.

lan

.

lme, in vitro veya in

ir (Till J.E, 1980;

Weissman, 2000). briyonik gel

ler.

pota lar (Daley ve ark., 2003).

2.2. lerin

kendini kendini yenileme, ve

yetenekleridir (Smith, 2001).

r am

(Fortier, 2005). Difera

bir

veya

ebilme yeteneklerine denir.

bir rine r. Kendini

yenileme (Self renewal): k in f

a

3

K kenler

Bunlar; b ,

iyonik ortam vb.) gibi etkenlerdir. Bu etkenler k

crel

ve uyaran etk i e

a irli

mesi

(Martin-Rendon ve Watt, 2003).

r

.

2.3 lerin

kaynaklardan elde edilebilmektedirler

ler 1).

1.

4

2.3.1.1. Embriyonik K

kendini yenileme yeteneklerine sahiptirler

(Martin, 1981). ken

cre tiplerine erken embriyonik

embriyo e

verir (Wobus, 2001).

2.3.1.2. Embriyonik Olmayan

bulunur l bilirler. Embriyolardan ve fetuslardan

elde e

(Hui ve ark., 2011).

multipotenttir. Bu c

, 2003).

2.3.1.2.1. Hematopoetik

da kan

u

lerinin KH)

memelilerde, HKH'ler a nde

bulunur. Bu nedenle,

HKH

zamanda k

lar (Huang ve ark, 2007). HKH

CD45, CD34 ve CD14 ile

fa ir (Dieterlen- e ve ark., 2002).

5

2.3.1.2.2. Mezenkimal (Stromal)

lk MKH enger ve ark. Kemik

ka n (Friedenstein

ve ark., 1976). H

Alman patolog Cohnheim (Prockop, 1997).

MKH r % 0.001-0.01'i

(Pittenger ve ark., 1999).

2008 s

;

0, CD71, CD44, Stro-1 gibi hematopoetik

olmayan h CD34, CD14, CD45, CD79

veya CD19 ve HLA-DR gibi tipik hema

,

sahip o

(Dominici ve ark., 2006).

MKH'ler fus Ortak bir MKH

ken, osteoblastlar matris biriktir

oidal olabilir (Grigoriadis ve ark., 1988). lerinin

integrinlerin, kadherinlerin kliklerden

kaynakla (Gumbiner, 1996).

6

2. Multipotent M (Murrell WD ve ark., 2015)

M

tra eri

ese edilen R

(RUNX2)

eksprese edilir ve ost -aktive

resept PAR-

rleridir (J.Marie, 2008).

3. MKH'l

7

2.3.1.2.3. eler

herhangi bir ,

tamir etmektir .

2.3

tenekleri

olabilir.

2.3.2.1. Totipo

m

blastomerler

(Brink ve ark., 2008).

2.3.2.2. Pluripotent

F olan

"blastosist"

endoderm, mezoderm ve ekdodermden gel it ine

bilirler. i er denir.

r. k

pik olarak yer

(Herzog ve ark., 2003).

2.3.2.3. Multipotent

ve

ler

buna iyi birer ebilen

8

uygun ektedir

(Jiang ve ark., 2002).

2.4. Vinposetin

Vinposetin (14-etoksikarbonil-(3a,16a-etil)-14,15-eburnamin),

inden elde edilen bir alkaloid olan vinkaminin sentetik bir

(Zhang ve ark., 2018).

il 4. Vinkamin ve vinposetinin Zhang ve ark., 2018)

Vinpos .,

geli ve

pazarlan . Vinposetin, piyasaya girmesinden bu yana;

A lerinde

(Zhang ve ark., 2018). v

yan etki ve toksisite bildirilmemi u

.

2.4.1

Emilim; vinposetin gastrointestinal sistemden

ilde emilir (Vereczkey, 1985). Vinposetin, or

uygulamadan sonra kan-beyin bariyerini g

. S

20'dir. rde ilk z

insandaki oral biyo % 7)

1984). Lohman ve maksimum

9

plazma konsantrasyonunu (Cmax yecekle

-100 daha

(Lohmann ve ark., 1992). Vinposetinin 10 mg oral

-1.5 saat sonraki maksimum plazma konsantrasyonunun 20-62 ng/ml

1979).

m; vinposetinin plazma proteinlerine % 85-

( 1985).

Metabolizma; Vinposetin CYP2C9 ile metabolize edilmektedir (Kong ve ark., 2016).

yum e lukla ana

metaboliti olan cis-apovinkaminik aside .

metabolitleri hidroksivinposetin, hidroksi-apovincaminik asit, dihidroksi-vinposetin-

gli (L.Vereczkey, 1985).

-2 saa

nde elimine edilir. Bu nedenle defa oral olarak 5-

10 mg olarak (J

plam plazma /saat

(L.Vereczkey, 1985).

2.4.2. Vinposetinin

2.4.2.1. Fosfodiesteraz 1 (PDE1)

Fosfodiesterazlar (PDE); siklik adenozin monofosfat (cAMP) ve siklik guanozin

(cGMP) a aktif olmayan -adenozin monofosfat ( -AMP) ve -

guanozin monofosfata ( -GMP) katalize ederek sinyalinde

yan enzimledir. PDE ne

. B

Hemen hemen memeli

bir fosfohidrolaz enzim ailesidir. PDE'ler

lmektedir (Lugnier, 2006).

Kalmodulin PDE1 katalitik aktivitesi, kalsiyum . Bu nedenle

PDE1; Ca2+/k . Vinposetin

(Ahn ve ark., 1997).

10

Vinposetinin PDE1A veya PDE1B IC50 ler olarak 8-20

iken, PDE1C IC50 40-50 olarak bildirilmektedir (Tablo 1). Bu

nedenle, vinposetin PDE1A/1B'ye

(Loughney ve ark., 1996; Yan ve ark., 1996).

2.4.2.2. Voltaj B + Kanal ibisyonu

Vinposetinin Na+ nda da i

Vinposetinin + IC50 10-50 M . Y

vinposetinin voltaj Na+

fenitoin kadar inhibe etti .

+ sel iskemi

i oldu Bunlar aras +

kanal blokerleri tetrodotoks olan riluzol ve Na+

kanallar (Urenjak ve

Obrenovitch, 1996).

2.4.2.3. Kinaz (IKK) syonu

Protein kinaz kompleksi (IKK), enflamatuvar tepkilere k

bir rol oynar. Harici enflamatuvar uyaranlara cevap olarak, bir dizi IKK kompleksi

aktive edilir. IKK; NF- B

sitokinler, kemokinler ve var

spresyonundan sorumlu olan anahtar bir transkripsiy

(Rothwarf ve ark., 1999). - B sitoplazmada

bulunur. NF-

kip eder. Sitokinl -

aktive eder (Mercurio ve ark., 1997). var sinyal, IKK

kinaz ile aktive olur. - B daha

nflamatuv in promot

u

, takiben NF-

NF- var mol neden olur

5) (Jeon ve ark., 2010). da ise vinposetinin NF- B ve

alfa (TNF- ) iskemi-

11

sonra enflamatuv (Wang ve ark., 2014). Vinposetinin

kinaz 50 d

ark., 2010).

' - v

nar. Bu nedenle vinposetin, IKK aktivitesini inhibe

ederek antiinflamatuv rebilmektedir (Jeon ve ark., 2010).

da vinposetinin makrofajlarda NF- B aktivasyonunu ve NF- B

ort Miyazawa ve ark., 2015). 2017

da olan 60 hastada

; vinposetinin akut iskemik inmeden sonra klinik

du u (Zhang ve ark.,

2018).

Tablo 1. IC50 (Zhang ve ark., 2018)

Hedefler IC50

PDE PDE1A / 1B: 8-20

PDE1C: 40 50

Beyin: PDE1A, 1B, 1C

Kalp: PDE1A, 1C

Makrofajlar: PDE1B

Voltaj

Na +

10 50 Beyin, kalp, damarlar

IKK

12

5. vinposetinin etkisini g ermektedir. Vinposetin IKK alt

lar

inflamasyonda rol oynayan TNF- - -8 gibi sitokin ve ayr kemokinlerin

ve ark., 2016).

2.4.3. Vinposetinin Etkileri

2.4.3.1.

t

k la nootrop

Patyar ve ark., 2011).

, vinposetinin iskemi koruyucu etkilerini ortaya

koymaktad (Jincai ve ark., 2014; , 2002).

Vinposetin beyin damar ci koz ve oksijen

ve ark., 2005). Vinposetinin ca bi e

bir antioksidan olarak i de (Deshmukh ve ark., 2009). Akut

b a (Zhang ve ark.,

2016). ro

13

gibi

k plastisite

in bir

adayl . if

elleri de Chen ve ark., 2007).

2.4.3.2 ar

sistemi hem ateroskleroz hem de iskemik inme de lezyon

Vinposetin endotel ve ni,

ve mikroglia IKK/NF- B yo mak suretiyle tuv

sa e ederek etkilemektedir.

vi

ederek ano neointimal

hiperplazi ve patolojik hafifletmede ortaya

(Zhang ve ark., 2015). hasar

vask ler trombo (Cai ve ark., 2012). Vinposetinin makrofajlarda lipit

birikim (Cai ve ark., 2013) kaslarda

r . Y

vinposetinin crelerinde osteoblastik farkl

(Ma ve ark., 2016).

2.4.3.3.

Ya ile vinposetinin kardiyak hipertrofi, fibroz k

Vinposetin miyosit yi,

fibroblast aktivasyon

PDE1 antagonize etmektedir (Wu ve

ark., 2017; Zhang ve ark, 2018).

kardiyomiyosit hipertrofisini (Miller ve ark., 2009).

2.5. Osteoporoz

Osteoporo

r (Kanis ve ark., 1994). 2006 ve 2007

da

14

milyondan fazla osteoporotik bildirilmektedir (Johnell ve

Kanis, 2006). riski

arda % 40-50, erkeklerde ise % 13 (Johnell ve Kanis, 2005). Bu

orunu .

le birlikte

stermektedir (Moerman ve ark., 2004; Li ve ark., 2011).

(RANK) /

morfogenetik proteini (BMP),

i olan osteoprotegerin (OPG) gibi

. RANKL, k osteoklast aktive

edici olarak RANKL-

07).

2.5.1. Osteoporoz ve K H

Osteoporoz ge

ka

MKH'lerin

il itlere veya osteoblastlara

; RUNX2 ve osteriks gi ,

adip a geni ile d zenlenir (J.Marie, 2008; Zhang X ve ark.,

2006).

ve k

genler COL1A1 ve COL1

en

(Kaneto CM ve ark., 2014).

, mineral birikimine yol a yerine

15

ineralizasyon

i artarken

b ve ark., 2007).

er klinikte ortopedide ost

defektlerini

ar insan

ler t osteoporotik

verilmesi ile arda anjiogenez ve osteogenez yoluyla osteoblast

(Qi ve ark., 2016).

k

er

(telomer nasyonu, onkojenik

transformasyon, v.b.)

ml

elerin yeni bir tedavi aja rmalar

16

3.

bi Gen

zi nde bulunan alet ve cihazlara ait bilgileri Tablo 2 de, sarf malzemelere

ait listeyi ise Tablo 3 de bulabilirsiniz.

Tablo 2. r

Marka (Laboratuvar,

Makine- at vb.)

M

Projede Kull

-80 Derin dondurucular 720 Lt

(Sanyo Panasonic VIP/ Binder)

G

,

- 20 Derin dondurucular

(Sanyo Biomedical Freezer) G Sarf malzemelerin,

numunelerin saklanm

(MVE 500 Series)

, ) ,

Sarf malzemelerin

CO2 G

Steril Kabin LaminAir Class II

(CleanAir)

Spektrofotometre (Multiplate Reader

PromegaGlomax)

testinde

Kar-

Santrif Farmakoloji,

Leika DMI 3000 Microskop lerin

Thermocycler (Biomac C1000 touch) Gen ekspresyon

(RocheLigthcycler 480)

Gen ekspresyon

Nanodrop (Shimadzu Biotech) RNA miktar tayini

ThermoMixer F1.5 RNA izolasyonunda

17

III)

karakterizasyonu,

apoptoz tayini

Otoklav Sterilizasyon

Mikropipetler ji

Bilgisayar akoloji

Tablo 3 sarf malzemeler

Malzeme

DMEM-Low glucose (Sigma) Besiyeri

Fetal Bovin Serum (Thermo) Besiyeri

Glutamax Besiyeri bil

Penicillin-Streptomycin (Thermo)

Trypsin-EDTA (Thermo) eleri pasajlamada

k f

Sitotoksisite, osteoj ,

gen ekspresyon r

5 ml (BD Biosciences)

nu (BD Biosciences)

MTT (Sigma)

Dulbecco fosfat tamponlu salin (DPBS)

(Biowest)

ler

E leri 1.5 ml, 2 ml

aka (RocheLigthcycler 480 ile

uy

TrizolReagent (Transgenbiotech)

PE anti-rat CD45 Antibody (Biolegend) MKH Karakterizasyonu

FITC anti-rat CD90/mouse CD90.1 (Thy-1.1)

Antibody (Biolegend)

MKH Karakterizasyonu

Purified Mouse Anti-Rat CD73, (BD

)

MKH Karakterizasyonu

Goat Anti-Mouse Ig FITC ( ) MKH Karakterizasyonu

Anti-CD79b antibody [AT107-2] (FITC)

(Abcam)

MKH Karakterizasyonu

Anti-CD105 antibody [SN6] (Abcam) MKH Karakterizasyonu

Anti-CD34 antibody [ICO-115]-PE ( Abcam) MKH Karakterizasyonu

Alizarin Red S (Fluorochem)

Oil O Red (Sigma) boyamak

RUNX2 Taqman qRT-PCR

18

ID: Rn01512298_m1)

Alkalen fosfataz (ALP) Taqman gen assay

(Thermo ID: Rn01516028_m1)

qRT-PCR

COL1A1

ID: Rn01463848_m1)

qRT-PCR

Beta aktin (

Rn00667869_ml)

qRT-PCR

Master mix (Thermo) qRT-PCR

cDNA sentez kiti (Thermo) cDNA sentezi

Liquid Substrate System solution

(Sigma)

Alkalen fosfataz boyama

% 100 ) RNA izolasyonu

Etanol (Sigma) RNA izolasyonu

Kloroform (Merck) RNA izolasyonu

Formaldehit (Sigma)

Beta-Galactosidase Staining Kit (Biovision) Senesens Testi

nylon syringe filters (GVS) Filtreleme

Kriyovial steril, 2 ml (Isolab) urma

Vinposetin ( )

Annexin V Apoptosis Detection Kit with 7-

AAD (Biolegend)

Apoptoz tayini

-Gliserofosfat disodyum tuzu hidrat (Sigma)

L-Askorbik asit (Sigma)

Deksametazon (Sigma)

r (Sigma)

3- -metilksantin (Sigma)

(Sigma)

3.2.

MKH r daha

-

315S176) elde Genom ve Merkezi ndeki -196

deki a muhafaza edilen 3. pasajdaki Sprague Dawley

3.2.1. Besiy

10 FBS, % 1 penisilin-streptomisin, % 1 glutamax

(DMEM)-LG kulla

19

besiyerleri taze olarak

Osteojenik Besiyeri: Beta-gliserofosfat 10 mM, askorbik asit 50 ,

deksametazon 0.1 10 FBS, % 1 glutamax ve %

1 penisilin-s -LG (Cho ve ark., 2010;

Colter ve ark., 2001; Delo ve ark., 2006).

Adipojenik Besiyeri: 3-izobutil-1-metilksantin 0.5 mM,

indometazin 60 , deksametazon 1 10 FBS, %

1 glutamax ve % 1 penisilin- -LG

(Zebisch ve ark., 2012; Dang ve ark., 2002; Sakaguchi ve ark., 2005).

3.2.2. ,

37 2

edilen k flasklarda 60-70

besiyeri arak ve % -EDTA eklenerek

l erek 1400 rpm

de 5 dakika besiyeri ile

seyr arak,

uygunluktaki flasklara

deneylerin ileriki her bir kriyovialde 1x106/ml

, % 10 dimetil (DMSO) eklenerek

uygulanarak -196

3.2.3. Karakterizasyonu

3.2.3.1.

in -

beklenir

.

20

MKH'lerin karakterizasyonunda r olan CD90, CD73 ve

CD79, CD34 ve CD45 n uygun antikorlar ile

. T175'lik f 6 adet

3x105 (Tablo 4).

30 dk 5 dk 300 g de

eklenerek 5 dk 300 g erek y 4. 5. ve

antikor 5 dk 300

g edilerek belirtilen primer antikor

. DPBS eklenerek BD FacsAriaIII cihazda

okutul

Tablo 4. antikorlar ve

1. Otofloresan (sadece

2. Primer antikor CD45 PE (0.4 ) + Primer antikor CD90 FITC (2.5 )

3. Primer antikor CD34 PE (1 ) + Primer antikor CD79 FITC (2.5 )

4. Primer antikor CD73 (2 ) + Sekonder antikor FITC (2 )

5. Primer antikor CD 105 (3 ) + Sekonder antikor FITC (3 )

6. Sekonder antikor FITC (3 )

3.2.3.2. sal Boyama

24 kuyucuklu plaka 1x104 MKH

60- ri

adipojenik ve leri ile 14

besiyerlerini

boyama ma, A

boyama

Oil O Red boya 3.2.7.

21

maddesinde ile

10 FBS, % 1 penisilin-streptomisin ve % 1

glutamax -LG uygu

3.2.4. a ; MTT Profilerasyon Testi

96 kuyucuklu plakalar

x103 MKH ekimi

vinposetin u 24. 48. ve 72. . Vinposetinin

plazma konsantrasyonu lite 20-62 ng/ml olarak

60 ng/ml vinposetin , 350.454 g/mol

plazma konsantrasyonu Vinposetinin

0.17, 1, 5, 10, 20, 25, 30, 40, 50 ve 100 M 96 kuyucuklu plakalara

. vinposetinin 25, 30 ve 40

M vinposetin konsantrasyon olan % 0.1, % 0.125 ve % 1.5 DMSO

sonraki 24, 48 ve 72.

saatlerde 3-[4,5-dimetiltiyazol-2-il]-

sonunda MTT

llerine mikroskopta

100 l

spektrofotometrede (Multiplate Reader Promega Glomax) 560 nm .

Elde edilen a

kontrol grubuna

ndiril

yani MKH'ler

belirlen

: Absorbans (vinposetin) x 100

Absorbans (kontrol)

3.2.5. Annexin V/7-ADD ile Apoptoz Testi

Apoptoz Annexin V-FITC/7-AAD apoptoz

belirleme kiti (FITC Annexin V Apoptosis Detection Kit with 7-AAD, Biolegend)

22

Annexin V, kalsiyum bir fosfatidilserin

proteinlerden annexin ailesinin bir ir. Fosfatidilserin normalde sadece

plazma bulunur, ancak erken apoptoz

zar asimetrisi kaybolur ve fosfatidilserin Florokrom

Annexin V ise apoptotik spesifik olarak abilir. Ancak

Annexin V apoptotik ve nekrotik yapamaz, bu nedenle 7-

amino-aktinomisin D (7-AAD) ile birlikte . 6 kuyucuklu

plakalar kuyucuk .5x105 MKH e de nemli ortamda

% 5 CO2 nemli

kuyucuklar; kontrol (sadece besiyeri), 0.17 M, 5 M ve 20 M

a takip eden 36. saatin sonunda

bir kez arak, tripsinize edilip 5 dk

1200 rpm sonra

ba eklendikten sonra

de hale getiril . Daha sonra FITC konjuge Annexin V antikoru ve 7-ADD

Annexin/7-

a ait sitometresinde BD FacsAria

III ile analiz edil . (Annexin negatif /7-ADD negatif), apoptotik

(Annexin pozitif /7-ADD negatif) ve nekrotik ler (Annexin pozitif/7-

ADD pozitif) BD FASCDiva Software le belirlen .

3.2.6. Betagalaktozidaz Boyama Kiti ile Senesens Tayini

de vinposetinin senesens

Beta-Galactosidase Staining Kiti (Biovision) 24 kuyucuklu

plakalar 1x104 MKH ekimi 5 CO2 hava

ontrol (sadece besiyeri),

0.17 M, 5 M ve 20 M vinposetin 24

besiyeri

PBS ile

bulunan fiks

edil erek ve CO2 C'deki

23

erek mavi-

renk zle .

3.2.7. yasal Boyama

24 kuyucuklu iki adet plaka (osteojenik

) x104 MKH una (kontrol,

ve ) ekim

. MKH'ler kuyucuk 60- taze

osteojenik besiyeri uygulanarak .

Kontrol grubuna

rubuna sadece osteojenik besiyeri uygulan

ve 20

konsan ilgili kuyucuklara eklenerek (Tablo 5), 37 deki %

5 CO2 n hava ile nemli ortamda

Tablo 5.

Gruplar Osteojenik

1. Kontrol: % 10 FBS, % 1 penisilin-streptomisin, %

DMEM-LG

2. Osteojen

3. V 0.17: O

4. V 5: O

5. V 20: O

ek ara ile yani 4., 7., 10., ve

13.

vinposetinin o

16. ve 19. g

21. g de vinposetinin

osteojenik sini incelemek

mikroskopta her kuyucuktan rastgele lar ekil daha sonra

I ile analiz edil .

24

3.2.7.1. Alizarin Red S Boyama

Alizarin Red S (Fluorochem 242552) kalsiyum ile reaksiyona girerek kalsiyum

Kuyucuklardaki besiyeri uzakla

15 dk %

Red S

lenerek oda 1 e sonunda boya

ne devam

.

3.2.7.2. ALP Boyama

ALP -5-Bromo-4-kloro-3-indolil fosfat

(BCIP)/NitroblueTetrazolium (NBT) substrat sistemleri, ALP enziminin

mavi-mor renkte

k

15 dk %

ALP-BCIP/NBT

eklenerek (Sigma B1911) r.

10 a kez distile su ile

lar .

3.2.7.3. Oil O Red Boyama

imyasal ed

boyama

an 15 dk %

P

. Bekleme

O R Oil O Red: distile su;

Oil O R

distile su ile

o

25

3.2.7.4. Image J P Analiz

Alizarin Red S ve ALP kantitatif

I version 1.5.

fot

6 a

olmayan alanlar ise .

6. Alizarin Red S boya

m), B:

rin

- Image-Type-8- 6.B).

- Siyah beyaz formata Image-Adjust-

Wand tool veya freehand selections 6.C).

- Analyze-Tools-

butonu ile eklen

-B (Area) Results penceresi Verilerin %

;

x 100

Toplam alan (5038848)

26

3.2.8. Gen Ekspresyon

lg

Elde edilen her bir gruba ait

G

polimeraz zincir reaksiyonu (RT-PCR) cihaz kullanarak RUNX2, ALP ve COL1A1

12 kuyucuklu iki

p a (osteojenik ) 3x104 MKH

ekim . MKH'ler kuyucuk taban 60-

.

grup kontrol, kontrol

, vinposetin ve (Tablo 6).

b di ve 2 7. 10. 13. 16. ve 19.

bir plakadan 14. dan ise de

total

Tablo 6. Gen ekspre

Gruplar

1. Kontrol: % 10 FBS, % 1 penisilin-streptomisin, %

DMEM-LG

2. 10 FBS, % 1

glutamax ve % 1 penisilin-s -LG

3. V 0.17: O

4. V 5: O

5. V 20: O

3.2.8.1.

trizol (Transgenbiotech .

kuyucuklardaki PBS ile bir kez , 500 trizol

eklen 7 e kimyasal lizis sonucu kuyucuk

arak, 5 dk oda bekletil .

Kloroform eklenerek bekletildikten sonra

10.000

27

katmanda i

Daha sonra izopropanol eklenerek .

beklemenin tir

dikk arak, (v/v) eklenip

kuvvetlice kar . 7500 erek s

. 5

dk sonunda bulunan n su eklenerek PCR (Thermo Mixer

F1.5) -

nanodrop (Shimadzu ve 260/280 nm, ve

260/230 nm Elde -80

7. Vahed ve ark, 2016)

28

3.2.8.2. cDNA Sentezi

Elde edilen RNA dan cDNA sentezlemek cDNA sentez kiti (Thermo High-

Capacity RNA-to- . Reaksiyonlara 600-800 ng/ml total RNA

. Her numuneden RNA ile reaksiyona girilebilmesi

olan RNA eyecek

, su, tampon ve enzim miktar

arak;

- H ,

- K bulunan enmi ir,

- RNA eklen ,

- Daha sonra isinde bulunan eklenerek pipetaj

l .

- aktivasyon

a basa ,

durmaya ayarlan

-20

3.2.8.3. -Zama Polimeraz Zincir Reaksiyonu (qRT-PCR)

o li genler olan RUNX2, ALP

ve COL1A1, referans (housekeeping) gen olarak da B-aktin kull larak gen ekspresyon

seviyelerine qRT-PCR Roche Ligthcycler 480

toplam hacim 20 e; genler, master mix

(Thermo Fisher Sci.) 1:10:5 l oran

k PCR

16 ld

n bir

l eklenerek Pre-

95 'de 10 sn, 60 'de 30 sn, 72 'de 1 sn 45

d 30 sn (threshold cycle)

29

RUNX2, ALP ve COL1A1 mRNA ekspresyon Beta-aktin

-

gibi hesaplanm

Kontrol Ct -aktin),

-

-

- (Livak ve ark.,

2001).

3.3. Analiz

lar ve mRNA gen ekspresyon deneylerinden

verilerin istatistiksel IBM SPSS Versiyon 16.0

istatistiksel paket . ndaki tek

varyans analizi ANOVA

testi ile, normalitesi ise Shapiro-Wilk testleri ile analiz edilerek;

esti, homojen olmayan gruplarda ise

post hoc Veriler aritmetik ortalama

(S.H.) . P<0.05 istatiksel olarak

Grafikler ise GraphPad Prism 8 l

30

4. BULGULAR

4.1. Karakterizasyonu

4.1.1. MKH'lerin Y B

de negatif be , ,

% 0 ve ise % 0.2 ve P

, ve 8 deki

tedir. killeri itibari ile hem de

a sitometrisi na MKH ait

iriz.

31

ekil 8. i MKH

A: otofloresan FITC na ait, B: otofloresan PE na

ait, C: CD79, D: CD45, E: CD34, F: CD73, G: CD105, H: CD90.

4.1.2. Boyamalar

e yine osteojenik ve

besiyerleri Oil O R

boyanan ; a ger ni

9).

% 0.2 % 0 % 0.2

% 91 % 100 % 84.6

% 0.8 % 0.6

32

9. MKH'lerin a lerinin ed ile boyamaya ait

; A: kontrol grubu B: Adipo m besiyeri

uygulanan grup (10x-B ; 50um)

Alizarin Red S boyama ile kals erinin ve ALP

boyama ile ALP enziminin mor renkte osteojenik

o

ekil 10).

10. M Alizarin red S ve ALP

boyamaya ait ; A: Kontrol grubu, B: O

besiyeri uygulanan gruba ait Alizarin Red S boyama, C: O besiyeri

uygulanan gruba ait ALP boyama (10x- ; 50um).

4.2. MTT Testi

Vinposetin uygulanan gruplarda 24. saatte sadece 100 vinposetin grubu ile kontrol

alma olmu

bir g

33

72. saatte ise 0.17-30 da kontrol

grubu istatistiksel ola ol tur (p<0.05) (Tablo 7).

h , 5 ve

vinposetin konsantrasyonlar ( 11).

DMSO 25, 30 ve vinposetin konsantrasyonlar daki ras la % 0.1,

% 0.125 ve % 0.15 . Bu oranlarda DMSO uygulanan gruplardaki

ise kontrol grubuna (p>0.05). Bu nedenle

konsantrasyonlardaki DMSO % 0.1 n

k olu

Tablo 7. Kontrol, vinposetin 0.17, 1, 5, 10, 15, 20, 25, ve DMSO

(% 0.1, % 0.125, % 0.15) uygulanan MKH'lere ait 24. 48. ve 72. MTT

aritmetik ortalama Standart hata (S.H.) olarak p ile birlikte

Gruplar 24. saat % c

48. saat % c

.

72. saat % can

Kontrol 100 100 100

Vin 0.17

86. 108.66

162.

Vin 1 96. 107.37 163.

Vin 5 103. (p:1) 114.97 89) 145.9

Vin 10 95. 113.15 59 (p:0.997) 174.

Vin 15 102. 124.26 171.

Vin 20 99. 125.43 190. 62 (p:0.00)

Vin 25 86.88 3.74 (p:0.992) 125.34 208.

Vin 30 94. 120.20 159.

Vin 40 92. 122.95 125.

Vin 50 73. (p:0.445) 112.85 :0.997) 107.

Vin 100

49. 90.99 1)

109.

DMSO

(% 0.1)

100. 117.21

109.

DMSO

(% 0.125)

94. 126.49

117. .30 (p:0.992)

DMSO

(% 0.15)

85. 103.92

116. (p:0.995)

34

35

11. Kontrol, vinposetin ve DMSO

(% 0.1, % 0.125, % 0.15) uygulanan MKH'lere ait 24., 48. ve 72. MTT testi %

edilen verilerin

idir. A post hoc Tukey testi ile ya l . *p < 0.05

fade etmektedir.

4.3. Apoptoz Testi Verileri

Vinposetinin

ap kontrol grubundan

nmemektedir ( 12).

36

12. Kontrol, 0.1

-AAD) testi l .

Q1: g pt Annexin- /7-ADD+), Q2: (Annexin+ / 7-

ADD+), Q3: nnexin - / 7-ADD- ) , Q4: apoptotik (Annexin + / 7-ADD-

)

4.4. Senesens Analizi

Vinposetin ve santrasyonlarda uyguland nda MK de

senesense - e ( 3).

13. Betagalaktozidaz MKH'lere ait invert

20x, B 50 ); A: Kontrol grubu, B: Vinposetin

0.17 , C: Vinposetin 5 , D: Vinposetin 20 konsantrasyon uygulanan gruplar.

37

4.5. Boyamalar

kontrol grubunda nkli boyan

edir. ve vinposetin

uygulanan kuyucuklarda boya mektedir. 21. e ait

k boya alanlar 4 ve 16).

da da kontrol grubunda

renk vinposetin uygulanan kuyucuklarda

ya 14. g e ait g erdeki boya ile

daha fazla bo 5 ve 17).

14. MKH'lerin

uygulanan), vinposetin 0.17 (V 0

uygulanan gruplara ait A- 14. ve B- deki Alizarin Red S

bo dir.

38

15. MKH'lerin kontrol, kontrol d

uygulanan), vinpos

- 14. ve B-

nt

1 Alizarin R ve 20

besiyeri uygulanan kontrol

alan ise sadece

anlam %

istatistiksel olarak le

vinposetin 5 ve konsantrasyonlarda ol

grubuna

(p<0.05) . Bu gruplara ait veriler ise

39

il 16. lere ait Alizarin Red S boyamalar

invert mikroskop (10x, ; 50 ) . A: 14. 14.

, C: 14. vinposetin 0.17 M, D: 14. vinposetin 5 M, E:

14. vinposetin 20 M, F: n kontrol, G: 21 , H: 21

vinposetin 0.17 M, I: 21 vinposetin 5 21 vinposetin 20 M

17. ALP boyamal e edilen invert

mikroskop (10X, ; 50 m) . A: 14.

, C: vinposetin 0.17 M, D: vinposetin 5 M, E: n

vinposetin 20 m, H: 21 vinposetin

0.17 M, I: 21 vinposetin 5 21 vinposetin 20 M

40

18. (sadece osteojenik

yeri uygulanan), vinposetin 5

r 14. ve 21. g lere ait

Alizarin Red S ve ALP boyama I

analizlerine ait rin ; A: n Alizarin Red

S, B: , C: Alizarin Red S, D: grafikleridir. malar

, her her bir kuyucuktan

erek analiz Veriler aritmetik ortalama standart hata olarak ifade

*p <0.05

kontrol

41

Tablo 8.

ait 14. ve 21

S.H. olarak, p d .

Gruplar

Alizarin Red S ALP

aritmetik

aritmetik

aritmetik

21.

aritmetik

46

Vinposetin

0. 7.65

(p:0.015) (p:0.245) (p:0.703) (p:0.161) Vinposetin

(p:0.001) (p:0.87)

.91

(p:0.979) (p:0.029)

Vinposetin (p:0.001) (p:0.033)

14.95 .73

(p:0.792) (p:0.006)

4.6. Gen Ekspresyon

O besiyeri uygulanmayan kontrol grubu, besiyeri

uygulanan grup ve 0.17 ve uygulanan

14. ve lerdeki RUNX2, ALP ve COL1A1 mRNA gen ekspresyon

seviyeleri analiz edil . 14. g kontrol grubu ile

a RUNX2, ALP ve COL1A1 mRNA gen

r. Ancak m besiyeri

uygulanan grup

(p>0.05). Vinposetin 0.17, 5 ve uygulanan RUNX2 gen

ekspresyon seviyesi gruba t ancak aza lar

(p>0.05). V 'da

ALP gen ekspresyon seviyesin ,

konsantrasyonlarda ise az , ksel olarak anla

ir (p>0.05). COL1A1 gen ekspresyonu ise vinposetinin

konsantrasyonlar da azalmakla birlikte bu azalma istatistiksel olarak anlam

(p>0.05) ( 19). Veriler Ta

42

19. MKH

vinposeti

RUNX2, ALP, COL1A1 nisbi mRNA gen ekspresyon

grafikleridir. A) RUNX2, B) ALP, C) COL1A1 mRNA nisbi gen ekspresyon

Veriler rlanan deneylerden, her seferinde

rlerinin orta 2-

rlerdir. iksel analiz t varyans

analizi (ANOVA) post hoc Tamhane T2 testi ile . #p<0.05 kontrol grubuna

43

Tablo 9. , kon , vinposetin

UNX2, ALP ve COL1A1 genlerine

ait 2- dir.

kez tekrarlanan deneylerden, her s k elde edilen Ct - . p1

2 ise kontrol olan ridir.

Gruplar Genler

RUNX2

ALP aritmetik or

COL1A1

Kontrol 1 1 1

Kontrol

(p1:0.24)

(p1:0.36)

(p1:0.63)

Vinposetin

2.6

(p1:0.43, p2:0.57)

(p1:0.81, p2:1)

(p1:0,02, p2:1)

Vinposetin

5 M

(p1:0.65, p2:0.53)

(p1:0.86, p2:0.95)

0

(p1:0.05, p2:0.80)

Vinposetin

(p1:0.41, p2:0.42)

(p1:0.23, p2:0.72)

(p1:0.29, p2:1)

izlerinde kontrol

besiyeri uygulanan (kontrol RUNX2 ve ALP mRNA gen

ekspresyon seviyelerinde a (p<0.05). COL1A1

m zeyinde ise azalma olmakla birlikte istatistiksel olarak

a (p>0.05). V RUNX2 ve

ALP mRNA gen ekspresyon lerinde gruba

istatiksel olarak . COL1A1 mRNA gen

ekspresyonu vinposetinin istatistiksel olarak

( ekil 20).

44

20. e kontrol, kontro

(V 2

21 UNX2, ALP, COL1A1 nisbi mRNA gen ekspresyon

grafikleridir. A) RUNX2, B) ALP, C) COL1A1 mRNA nisbi gen ekspresyon

Veriler her seferinde

arak elde edilen ala 2-

aritmetik erdir. varyans

analizi (ANOVA) ile yap post hoc; RUNX2 ve ALP , COL1A1

Tamhane T2 . #p<0.05 ,*p<0.05 ise kontrol

#

45

Tablo 10. M

vinposetin 0.17 21 UNX2, ALP ve

COL1A1 genlerine ait 2- yani 'nisbi mRNA gen ekspresyonu Veriler

rlanan deneylerde

n ort esaplanan 2-

Gruplar Genler

RUNX2

ALP S.H.

COL1A1 aritmetik

Kontrol 1 1 1

Kontrol

(p1:0.01)

(p1:0.03)

(p1:0.99)

Vinposetin

37

(p1:0.51, p2:0.87)

(p1:0.1, p2:0.21)

(p1:0.45, p2:0.938)

Vinposetin

(p1:0.60, p2:0.006)

(p1:0.41, p2:0.044)

(p1:0.73, p2:0.986)

Vinposetin

(p1:0.32, p2:0.013)

(p1:0.65, p2:0.022)

(p1:0.03, p2:0.788)

46

5.

olan RUNX2, homeodomain proteinleri ve osteriks bulunur. RUNX2 gen

kemik morfogenetik

beta ( ),

lar ve

erin osteojenik

-2 ve osteokalsin gen

karak

isinin

TNF- sistemi ji

me , ojenez li

patolojik olaylarda

-

osteoblast fonks por e .

TNF- -

doku ka - aktivasyonu

ile NF- -

F-

47

ve osteokalsin ekspr

inhibit rolidin- TNF-

Hess ve ark. (2009) yap -

iyeri uygu y -

-PCR, western blot analizi ve

o t r. TNF- P ve ALP gibi osteojenik

kspresyon yle

-

tutar TNF-

far a . Hess ve ark. (2009)

an -

n genlerin ekspre

ilmi . t olarak -

-7082 uygulanarak farelerd

a ise NF- , BMP-2 ekspresyonunu ve

aktivitesini inhibe ederek kondrojenezi ve kemik i bas

(Wu ve ark., 2007). NF-

in 5

n NF-

yumlu olarak

ta-gl

model larak vi

2016). Vinposetinin ALP,

osteokalsin, kollajen tip I, RUNX2 ve BMP-

rinin mi

dakika sonra ERK/

k; NF- p65'i

ost steri Ma ve ark., 2016).

48

Ma ve a

d 12. erlendirirken bizim

rine etkileri 14.

NF-

mevcuttur. n bir

aktive e

osterix ve kemik sialoproteinin ekspresyon

e ark., 2013). Kaneki ve

na

ark., 2006).

al mada Chen ve

inhibe edebile ini ve NF-

Bir IKK

- farmakolojik inh

-

ve bu nedenle NF- sta avisinde yeni bir

rdir (Chen ve ark., 2013).

Vinposetinin osteojenik

ermesinde vinposetinin mekanizmas PDE

inhibis . Vinposetin ,

e sahiptir (Ahn ve ark., 1997).

50 -20

50 -50 edenle,

vinposeti oughney ve ark.,

1996; Yan ve ark., 1996). Muhtemelen, PDE1'in inhibisyonu cAMP ve cGMP

trombositlere uygulanan etk

de PDE i

denbufilinin oral uygulama ile kemik

49

zerine etkilerinin da n

emi

g ster . PD

tedavisinde (Miyamoto ve ark., 1997). Bu

PDE4 inhibi pen mik

sini art

- ekspresyonunda bir

azalmaya neden olarak kemik me

Kinoshita ve ark., 2000).

un kemik

(Huyut ve ark., 2018).

lmakt r.

mevcuttur. Fare kemik li protein uygulan

ile ren bir mekanizma ile

osteoklastogenezin tarihinden

roid hormonun

ve ik etkilerinde, hem cAMP

ark., 1997; Carpio ve ark., 2001; Kawane ve ark., 2003). Wu ve ark. (2013),

uygulayarak

-

art ve .

lazer

Wu ve

r n

seviyesinin y teb .

Vinposetin ise PDE1 inhibisyonu etkisi sonucu cAMP hidrolizini 8-

konsantrasyonlarda inh aktad

50

bilgi rken

tedir..

Kinoshita cA NF-

ve IKK-NF-

a, ke

kapasitelerinin azalmas ositlere

MKH'lerin

de adipositlere k

ve ark.,

vo

shid ve Kwoh, 201

nin erin ost

bireylerdeki osteoporoz

Bununla birlikte, vinposetinin

in klini

durum in ellik talara vinposetin

an dikkat etmek gerekebilir.

51

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65

Uy rkiye (T.C)

16.07.1991 - Kayseri

Medeni Durum: Bekar

E-mail: yldrmesma_@hotmail.com

Sahabiye M Cad. Erciyes Sit. B blok A giri

Derece Kurum Mezuniyet Tarihi

-

Lisans 2015

Lise Kocasinan Anadolu Lisesi, Kayseri 2009

Kurum

2019-Halen T.C. -

2016-2019 3

YAYINLAR