本文關鍵詞： CD146~+血管周圍細胞 間充質干細胞 層流剪切力 細胞周期抑制 凋亡 微重力 軟骨誘導分化　出處：《華中科技大學》2012年博士論文　論文類型：學位論文

【摘要】：目的：1.分離、純化CD146+血管周圍細胞,并進行軟骨誘導,探討其成為軟骨修復組織工程的種子細胞可行性；進一步揭示間充質干細胞(MSCs)的血管周圍來源。2.研究層流剪切力(laminar shear stress; LSS)對體外培養(yǎng)的大鼠MSCs增殖和凋亡的影響；并初步探討其抗凋亡機制。3.在體外微重力條件下三維誘導大鼠MSCs向類髓核細胞分化,為退變椎間盤的修復提供種子細胞。方法：1.從大鼠脂肪組織中分離、純化CD146+血管周圍細胞；進行軟骨誘導；通過RT-PCR和Western blot檢測軟骨分化；進一步檢測CD146+血管周圍細胞的遷移能力。2.大鼠MSCs分組予以生理范圍內的層流剪切力力學干預。通過流式細胞學檢測細胞周期和凋亡；RT-PCR檢測各組細胞的Bcl-2、Bax凋亡相關基因的mRNA表達情況。3.在微重力條件下采用顆粒培養(yǎng)法(pellet culture)誘導骨髓MSCs向髓核細胞分化,建立三個細胞培養(yǎng)組：實驗組(0ng/ml TGF-β1和微重力),陽性對照組(10ng/ml TGF-β1和微重力),空白對照組(0ng/ml TGF-β1)。WST-8檢測細胞增殖；組織化學和RT-PCR檢測細胞分化。結果：1.CD146+血管周圍細胞成功從S-D大鼠脂肪組織中分離、純化培養(yǎng)；流式細胞檢測顯示：CD146陽性,CD45、CD56、CD34陰性；Sox9和Aggrecan在1mRNA和蛋白水平上均高于對照組；細胞遷移實驗顯示實驗組較高。2.在加載生理力度的LSS (15-dyne/cm2)后,MSCs的DNA合成明顯受到抑制；細胞周期檢測發(fā)現(xiàn)：在細胞加載LSS (15 dyne/cm2)后的不同時間點,處于S和G2/M期的細胞百分含量隨著時間變化有明顯的下降；細胞凋亡檢測顯示：,實驗組的活細胞比率由59.39%上升到82.37%,24小時保持在81.55%,實驗組的凋亡細胞比率由40.61%下降到17.49%,24小時保持在18.83%；生理范圍內的力學刺激促進抗凋亡基因的表達,即Bcl-2表達增加,Bcl-2/Bax比率增高。3.誘導培養(yǎng)第三天,TGF-βl對MSCs的增殖有輕度的促進作用；誘導培養(yǎng)第七天,組織染色顯示微重力下兩組都有膠原和蛋白聚糖存在；蛋白聚糖含量檢測實驗組顯著增高；RT-PCR結果顯示微重力條件下軟骨細胞標志基因(Sox-9,蛋白聚糖,Ⅱ型膠原)表達量增高；實驗組蛋白多糖與膠原的比率(proteoglycans/collagen)是陽性對照組的3.4倍,進一步提示MSC向髓核細胞方向分化。結論：1.成功分離、純化CD146+血管周圍細胞；誘導后CD146+血管周圍細胞向軟骨細胞方向分化；CD146+血管周圍細胞有較好的細胞遷移能力。2.生理范圍內的LSS引起MSCs細胞周期抑制和抗凋亡作用,介導MSCs的休眠,這有利于MSCs的干細胞特性保持。3.MSCs在微重力條件下自發(fā)向髓核細胞方向分化,證明三維立體培養(yǎng)是誘導MSCs向髓核細胞分化的條件之一,可為椎間盤退變性疾病的干細胞移植治療提供種子細胞。
[Abstract]:Objective to isolate and purify the perivascular cells of CD146 and to investigate the feasibility of becoming seed cells for cartilage repair tissue engineering. To further reveal the sources of mesenchymal stem cells (MSCs) around blood vessels. 2. To study laminar shear stress in laminar flow. Effects of LSS on proliferation and apoptosis of rat MSCs in vitro; The mechanism of anti-apoptosis was also discussed. 3. Three-dimensional differentiation of rat MSCs into nucleus pulposus cells was induced by microgravity in vitro. To provide seed cells for the repair of degenerated intervertebral disc. Methods: 1. Isolated and purified CD146 perivascular cells from adipose tissue of rats. Cartilage induction; Cartilage differentiation was detected by RT-PCR and Western blot. The migration ability of perivascular cells of CD146 was further measured. Rat MSCs was divided into two groups and subjected to laminar shear stress intervention in physiological range. Cell cycle and apoptosis were detected by flow cytology. Bcl-2 was detected by RT-PCR. Expression of Bax apoptosis-related gene mRNA. 3. Bone marrow MSCs was induced to differentiate into nucleus pulposus cells by granular culture under microgravity. Three cell culture groups were established: experimental group (0 ng / ml TGF- 尾 1 and microgravity), positive control group (10 ng / ml TGF- 尾 1 and microgravity). The cell proliferation was detected by TGF- 尾 _ (1) and WST-8 in the blank control group (0 ng / ml TGF- 尾 _ (1)). Results the perivascular cells were isolated and cultured successfully from the adipose tissue of S-D rats. Flow cytometry showed that CD146 was positive and CD45, CD56 and CD34 were negative. Sox9 and Aggrecan were significantly higher than those of the control group at 1 mRNA and protein levels. The cell migration assay showed that the DNA synthesis of LSS was significantly inhibited after the physiological stress of 15-dyne / cm ~ (2). Cell cycle analysis showed that the percentage content of cells in S and G _ 2 / M phase decreased significantly at different time points after cell loading with LSS 15 dyne / cm ~ (2). Cell apoptosis test showed that the percentage of living cells in the experimental group increased from 59.39% to 82.37 and remained at 81.55% for 24 hours. The percentage of apoptotic cells in the experimental group decreased from 40.61% to 17.49 and remained at 18.83 for 24 hours. Mechanical stimulation in physiological range promoted the expression of anti-apoptotic genes, that is, the expression of Bcl-2 increased, the ratio of Bcl-2 / Bax increased .3.The third day of induction culture. TGF- 尾 l slightly promoted the proliferation of MSCs. After 7th days of induction and culture, tissue staining showed the presence of collagen and proteoglycan in both groups under microgravity. The content of proteoglycan was significantly higher in the experimental group. RT-PCR results showed that the expression of chondrocyte marker gene (Sox-9, proteoglycan, type 鈪，

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