本文選題：軟骨調(diào)節(jié)素-I + 生物因子��； 參考：《大連醫(yī)科大學(xué)》2012年碩士論文

【摘要】：研究目的：探索ChM－Ⅰ的上調(diào)表達(dá)誘導(dǎo)MSCs向軟骨細(xì)胞方向分化的作用。 研究方法： 1.密度梯度離心法獲得大鼠MSCs，傳代培養(yǎng)并擴(kuò)增。使用流式細(xì)胞儀檢測(cè)細(xì)胞表面抗原（CD90、CD29）。將凍存的穩(wěn)轉(zhuǎn)pcDNA3.1（+）/ChM－Ⅰ質(zhì)粒的MSCs和穩(wěn)轉(zhuǎn)pcDNA3.1（+）質(zhì)粒的MSCs復(fù)蘇及擴(kuò)培。 2.高糖DMEM無(wú)血清特定培養(yǎng)誘導(dǎo)液（含10ng/mlTGF-β1、100nmol／L地塞米松、50umol／ml抗壞血酸、1％ITS） 3.實(shí)驗(yàn)分六組（1）：實(shí)驗(yàn)1組（穩(wěn)定轉(zhuǎn)染表達(dá)載體pcDNA3.1(+)/ChM-Ⅰ的MSCs細(xì)胞組）（2）：實(shí)驗(yàn)2組（穩(wěn)定轉(zhuǎn)染表達(dá)載體pcDNA3.1(+)/ChM-Ⅰ的MSCs細(xì)胞＋誘導(dǎo)液組）(3)：對(duì)照1組（正常MSCs組）(4)：對(duì)照2組（正常MSCs+誘導(dǎo)液組）(5)：對(duì)照3組（穩(wěn)定轉(zhuǎn)染表達(dá)載體pcDNA3.1（+）的MSCs細(xì)胞組）(6)：對(duì)照4組(穩(wěn)定轉(zhuǎn)染表達(dá)載體pcDNA3.1（+）的MSCs＋誘導(dǎo)液組),細(xì)胞培養(yǎng)至7天、14天、21天三個(gè)時(shí)間點(diǎn)，通過(guò)使用倒置顯微鏡觀(guān)察細(xì)胞形態(tài)的改變；甲苯胺藍(lán)染色檢測(cè)軟骨基質(zhì)的分泌情況；免疫細(xì)胞化學(xué)檢測(cè)軟骨特異性Ⅱ型膠原的表達(dá)情況。通過(guò)以上實(shí)驗(yàn)方法說(shuō)明表達(dá)ChM-Ⅰ的大鼠MSCs細(xì)胞株具有向軟骨細(xì)胞分化的能力。 研究結(jié)果： 1. MSCs細(xì)胞株的擴(kuò)增培養(yǎng)及鑒定： 單細(xì)胞懸液接種48h后換液，倒置顯微鏡觀(guān)察并拍照：可于培養(yǎng)皿底部見(jiàn)到呈梭形或三角形的貼壁細(xì)胞，培養(yǎng)11～13天后培養(yǎng)皿底部細(xì)胞密度達(dá)到85%以上。通過(guò)傳代，細(xì)胞在6h內(nèi)可達(dá)80%以上貼壁，，細(xì)胞生長(zhǎng)速度加快，可見(jiàn)細(xì)胞呈成纖維細(xì)胞樣生長(zhǎng)，折光性較強(qiáng)，群落呈魚(yú)群樣。流式細(xì)胞儀鑒定結(jié)果：MSCs陽(yáng)性表面抗原CD90、CD29的表達(dá)率分別為99.79%、98.41%；MSCs陰性表面抗原CD45、CD11b/c的表達(dá)率分別為2.77%、2.51%。提示MSCs達(dá)到實(shí)驗(yàn)要求。 2.凍存的穩(wěn)定轉(zhuǎn)染表達(dá)載體pcDNA3.1(+)/ChM-Ⅰ的MSCs細(xì)胞組和穩(wěn)定轉(zhuǎn)染表達(dá)載體pcDNA3.1（+）的MSCs細(xì)胞組復(fù)蘇及擴(kuò)培： 常規(guī)復(fù)蘇后，通過(guò)增值、傳代，細(xì)胞生長(zhǎng)旺盛，排列密集整齊，呈編織狀、旋渦狀。 3.六組細(xì)胞甲苯胺藍(lán)染色和Ⅱ型膠原免疫組化結(jié)果及分析：實(shí)驗(yàn)2組誘導(dǎo)后的細(xì)胞和3個(gè)對(duì)照組的細(xì)胞由梭形向多邊形轉(zhuǎn)變。甲苯胺藍(lán)染色和Ⅱ型膠原免疫組化結(jié)果顯示：對(duì)照1、3組檢測(cè)的特異性軟骨基質(zhì)的分泌和軟骨Ⅱ型膠原的表達(dá)均為陰性；實(shí)驗(yàn)1組和對(duì)照2、4組均檢測(cè)存在特異性軟骨基質(zhì)的分泌和軟骨型Ⅱ膠原的表達(dá)，但實(shí)驗(yàn)1組為弱陽(yáng)性，對(duì)照2、4組為陽(yáng)性，說(shuō)明ChM-Ⅰ的上調(diào)表達(dá)可誘導(dǎo)MSCs向軟骨細(xì)胞分化，但不及TGF-β1的效率；實(shí)驗(yàn)2組檢測(cè)軟骨基質(zhì)的分泌和軟骨Ⅱ型膠原的表達(dá)呈強(qiáng)陽(yáng)性，優(yōu)于對(duì)照2、4組；實(shí)驗(yàn)2組和實(shí)驗(yàn)1組均能檢測(cè)到特異性軟骨基質(zhì)的分泌和軟骨Ⅱ型膠原的表達(dá)，且實(shí)驗(yàn)2組優(yōu)于實(shí)驗(yàn)1組；說(shuō)明穩(wěn)定轉(zhuǎn)染表達(dá)載體pcDNA3.1(+)/ChM-Ⅰ的MSCs細(xì)胞在TGF-β1的協(xié)同作用下向軟骨細(xì)胞方向轉(zhuǎn)化的能力顯著提高。 研究結(jié)論： 1.本實(shí)驗(yàn)證明上調(diào)表達(dá)ChM-Ⅰ可誘導(dǎo)MSCs向軟骨細(xì)胞分化。 2.在TGF-β1的協(xié)同作用下，上調(diào)表達(dá)ChM-Ⅰ的MSCs可提高向軟骨細(xì)胞分化的能力。 3.多重表達(dá)載體和多種生物因子聯(lián)合誘導(dǎo)MSCs可能成為構(gòu)建組織工程軟骨的重要方式。
[Abstract]:Objective: To explore the effect of up regulation of the expression of MSCs on the differentiation of chondrocytes into chondrocytes.
Research methods:
The rat MSCs was obtained by 1. density gradient centrifugation. The cell surface antigen (CD90, CD29) was detected by flow cytometry. The MSCs of the frozen pcDNA3.1 (+) /ChM I plasmid and the MSCs recovery and expansion of the stable pcDNA3.1 (+) plasmid were obtained.
2. high glucose DMEM serum-free specific culture inducing liquid (containing 10ng/mlTGF- beta 1100nmol / L dexamethasone, 50umol / ml ascorbic acid, 1%ITS)
3. the experiment was divided into six groups (1): the 1 group (2) of the stable transfected expression vector pcDNA3.1 (+) /ChM- I (2): the 2 groups (3) of the MSCs cells of the stable transfection vector pcDNA3.1 (+) /ChM- I (3): the control 1 (normal MSCs group) (4): the control 2 group (5): the control 3 group (stable transfection expression vector pcD) NA3.1 (+) MSCs cell group) (6): control 4 groups (stably transfected expression vector pcDNA3.1 (+) MSCs + inducer group), cell culture to 7 days, 14 days, 21 days at three time points, by using inverted microscope to observe cell morphological changes; toluidine blue staining detection of cartilage matrix secretion; immunocytochemical detection of cartilage specificity The expression of type II collagen indicates that the MSCs cell line expressing ChM- I has the ability to differentiate into chondrocytes.
The results of the study:
Amplification and identification of 1. MSCs cell lines:
The single cell suspension was inoculated with 48h after inoculation, and the inverted microscope was observed and photographed: the cell density of the cell at the bottom of the culture dish could be seen at the bottom of the culture dish. The cell density at the bottom of the culture dish reached over 85% after 11~13 days. Through the passage, the cells could be more than 80% in the 6h, and the cell growth speed was quicker and the cells were fibroblasts. MSCs positive surface antigen CD90, CD29 expression rate was 99.79%, 98.41%, MSCs negative surface antigen CD45, CD11b/c expression rate was 2.77% respectively, 2.51%. suggesting MSCs reached the experimental requirements.
2. cryopreserved transfected expression vector pcDNA3.1 (+) /ChM- I MSCs cell group and stable transfection expression vector pcDNA3.1 (+) MSCs cell group resuscitation and expansion:
After regular resuscitation, the cells grew vigorously and arranged densely and neatly, and knitted and whirlpool shaped.
3. six groups of toluidine blue staining and type II collagen immunohistochemical results and analysis: the cells in the 2 groups and the 3 control groups were transformed from spindle to polygon. The results of toluidine blue staining and type II collagen immunohistochemical staining showed that the specific cartilage matrix secretion and cartilage type II collagen expression in the control group were detected in the control group 1,3. Both of the 1 groups and the control 2,4 groups detected the secretion of specific cartilage matrix and the expression of cartilage type II collagen, but the 1 groups were weak positive and the control group 2,4 was positive, indicating that the up-regulated expression of ChM- I could induce the differentiation of MSCs into chondrocytes, but not the efficiency of TGF- beta 1; the 2 groups of experiments detected the secretion and soft tissue of cartilage matrix. The expression of type II collagen was strongly positive, superior to the control group 2,4, and the 2 and 1 experimental groups were able to detect the secretion of the specific cartilage matrix and the expression of collagen type II collagen, and the 2 groups were superior to the experimental group 1, indicating that the stable transfected expression vector, pcDNA3.1 (+) /ChM- I, was directed to the cartilage cell side under the synergistic effect of TGF- beta 1. The ability to convert to transformation is significantly improved.
The conclusions are as follows:
1. this experiment showed that the expression of ChM- I could induce MSCs to differentiate into chondrocytes.
2. under the synergistic effect of TGF- beta 1, up regulation of ChM- I MSCs can enhance the ability to differentiate into chondrocytes.
3. the combined expression of multiple expression vectors and various biological factors can induce MSCs to become an important way to construct tissue-engineered cartilage.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R318.08

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