Induction of mice bone marrow derived mesenchymal stem cells differentiated into chondrocyte in vitro

العناوين الأخرى

تحفيز الخلايا الجذعية من نقي العظام في الفئران إلى خلايا غضروفية متمايزه في الزجاج

مقدم أطروحة جامعية

Salman, Marwah Ibrahim

مشرف أطروحة جامعية

Umran, Mahfuzah Abbas
al-Shammari, Ahmad Majid Hamzah

أعضاء اللجنة

Mutlak, Baydaa H.
Aziz, Ghazi Munim
Tawfiq, Amir T.

الجامعة

جامعة بغداد

الكلية

كلية العلوم

القسم الأكاديمي

قسم التقانة الإحيائية

دولة الجامعة

العراق

الدرجة العلمية

ماجستير

تاريخ الدرجة العلمية

2013

الملخص الإنجليزي

Mesenchymal Stem Cells (MSCs) are multipotent cells holding great potential in cell based therapy for regenerative medicine, and considers an attractive source for cartilage repair because of their availability to chodrogenic differentiation by progressing sequentially through the expression of cartilage specific extracellular matrix molecules, this requires well-defined and efficient protocols for directing the differentiation of stem cells into the chondrogenic lineage.

The study designed to evaluate the best method for isolation mesenchymal stem cells (MSCs) from bone marrow (BM) of albino mice according to three protocols for isolation, purification and proliferation of MSCs in vitro, afterward differentiation of these MSCs into chondrocytes.

These three protocols included classical protocol, gradient centrifugation by using lymphocyte separation media Ficoll and percoll.

Bone marrow aspirate were obtained from male mice at age 4-6 week and cultivated in minimal essential media (MEM) supplemented with 15% fetal bovine serum (FBS) in tissue culture flask.

The results demonstrated that the MSCs yield by using classic method were much higher than that obtained by the two other methods, the MSCs were subculture when the monolayer reached at 80-90% confluence.

The obtained MSCs were examined by phenotype analysis for positive CD markers, CD 105, CD 90, CD 44, and negative CD 34 and CD 45.The isolated of MSCs obtained from above steps were cultivated using three different culture methods included: 1.

Pellet (micro-mass) culture system.

2.

Two dimensional (2D) culture system (morphological study).

3.

Three dimensional (3D) matrix scaffold (polycaporolacton) (PCL).

Three type of chondrogenic differentiation media were used in each culture method, media type one consisting of DMEM high glucose(4.5g/L),LII glutamine supplemented with heps ,10ng/ml recombinant human transforming growth factor beta1(TGF-β1), 100nM dexamethasone, ascorbate2-phospho (37.5 μ/ ml), 10ng/ml recombinant murine insulin like growth factor 1(IGF1).

10ng/ml recombinant human fibroblast growth factor, basic,(FGFb).Type two of chondrogenic differentiation media consist of all composition of type 1 media except the absence of IGF-1, type three of chondrogenic differentiation media consist of all composition of type 1 media except the absence of TGF- β1.

In pellet culture system after 21 days of incubation the cell culture were assessed histological by hematoxilyine and eosine (H & E) and alcian blue.

Collagen type I (COL1A1), collagen type II (COL2A1) and cartilage acidic protein 1(CRTAC1) were assessed by immunohistochimistry.

In this method of culture the formation of distinguished cartilage was showed in media type1.

In 2D culture system (morphological study) the cells assessed by immunohistochimistry analysis after 7,14 and 21 days of incubation by using COL1A1 and COL2A1.

Negative results were saw after 7 days of incubation in all three type of media.

After 14 days weak positive results were seem just in media type 1.

After 21 days of incubation in media type 1 positive results were seem in COL1A1 ,weak positive in COL2A1, in type 2 media weak positive were seem in COL1A1, COL2A1 while in type 3 media negative result were appearance in the both type of chondrocyte markers.

Third model of culturing system of MSCs directed to chondrogenic differentiation by 3D poly-З-caprolacton (PCL) scaffold were examined histologically after 21 days of cultivation MSCs in three types of media.The histological sections of PCL containing adherent cells within scaffold showed positive staining of H&E of cartilage formation in 3 types of media while ,the staining by alican blue reveals positive staining of cartilage formation in sections of scaffolds contain MSC, cultivated in media 1, positive result for cartilage formation in media 2 and media 3, the detection of glycosaminoglycan (GAG) activity,COL1A1 and COL2A1 were done since IIIthese material consider as a markers for the modulating of MSCs to chondrocyte.

The results have led to widely held concept that polymers of biomaterials will be necessary and useful for cell therapy with MSCs derived to chondrocyte

التخصصات الرئيسية

الأحياء

عدد الصفحات

129

قائمة المحتويات

• APA
• MLA
• AMA

لغة النص

الإنجليزية

نوع البيانات

رسائل جامعية

رقم السجل

BIM-601196