本文選題：胎盤 + 間充質(zhì)干細胞　； 參考：《中國醫(yī)科大學(xué)》2008年碩士論文

【摘要】： 目的 間充質(zhì)干細胞(Mesenchymal stem cells,MSCs)是屬于中胚層的一類多能干細胞,主要存在于結(jié)締組織和器官間質(zhì)中,以骨髓組織中含量最為豐富。近年來,隨著人們對間充質(zhì)干細胞生物學(xué)特性及功能的深入研究,已成功地從人骨髓、外周血、肌肉、脂肪、臍血、羊水及胎兒組織中分離并鑒定出MSCs。胎盤作為胚胎發(fā)育中維系母體和胎兒氧氣及營養(yǎng)物質(zhì)交換的重要暫時性器官,由起源于細胞滋養(yǎng)層和胚外中胚層的胎兒叢密絨毛膜和母體子宮基蛻膜共同組成,無論從解剖結(jié)構(gòu)還是在發(fā)育行為上,都包含了較為幼稚的胚胎及趨于成熟的成體干細胞成分;胎盤在胎兒娩出后即完成使命,成為“廢棄”物,對其研究不會涉及倫理道德問題,因此目前已成為尋找人類間充質(zhì)干細胞新來源及提高臨床應(yīng)用效果的研究熱點。本文就人胎盤來源的間充質(zhì)干細胞(Placenta-derived Mesenchymal Stem Cells,PMSCs)體外分離和培養(yǎng)的方法及PMSCs在單層培養(yǎng)中向軟骨細胞定向分化的條件作以論述。 方法 1、人胎盤間充質(zhì)干細胞的提取 取足月剖宮產(chǎn)胎兒的胎盤,剝離子體蛻膜,剪取蛻膜下組織,PBS沖洗兩次,室溫靜置10min,將組織塊剪碎約1mm~3,均勻置于培養(yǎng)器底部,應(yīng)用10%DMEM培養(yǎng)基培養(yǎng)于37℃5%CO_2培養(yǎng)箱,每3d換液一次,待細胞沿培養(yǎng)器底部生長面積達80%～90%時,按常規(guī)方法傳代。 2、胎盤細胞表型測定 取第3代細胞,以0.25%胰蛋白酶消化3min后,調(diào)整細胞數(shù)為1×10~6/L,加入5μL小鼠血清封閉15min后,加入鼠抗人PE標(biāo)記的單克隆抗體,冰上避光反應(yīng)20min,PBS洗2次后,4℃1000r/min離心5min,棄上清后加入200μL冷PBS吹打混勻后,流式細胞儀檢測。 3、誘導(dǎo)分化 將第3代胎盤來源的間充質(zhì)干細胞胰酶消化后制成細胞懸液,離心棄上清后PBS洗3遍,分別以2×10~4/cm~2密度接種于預(yù)先以0.1%明膠涂抹的六孔細胞培養(yǎng)液中,待細胞貼壁后改換軟骨誘導(dǎo)劑,置于37℃5%CO_2培養(yǎng)箱中培養(yǎng),每3d換液一次。 4、甲苯胺藍染色 取培養(yǎng)21d的兩組細胞,PBS換2遍后,4%多聚甲醛固定15～30min,PBS洗3遍后,1%甲苯胺藍染色2～4h,95%酒精洗3遍后光鏡下觀察。 5、免疫細胞化學(xué)檢測Ⅱ型膠原表達 具體步驟按SABC染色試劑盒說明書操作,并棄孔板中培養(yǎng)液,PBS(0.02mol/LpH7.2～7.6)液洗3遍,4%多聚甲醛固定30min,0.3%H_2O_2滅活內(nèi)源性酶,BSA封閉后分別加入1:200稀釋的兔抗人-抗Ⅱ型膠原,4℃孵育過夜,用山羊抗兔-小鼠IgG孵育1h,DAB顯色劑顯色,蘇木精復(fù)染后光鏡下觀察。 結(jié)果 組織塊培養(yǎng)11d左右,可見少量細胞爬出,呈圓形或多角形,隨細胞數(shù)目增多,細胞呈旋渦狀生長,胞體變細長,形態(tài)類似成纖維細胞,培養(yǎng)3w左右,細胞可達80%融合,細胞傳代貼壁后,重新長成梭形細胞,該細胞傳至20代仍可穩(wěn)定生長。流式細胞儀檢測顯示,來源于人胎盤組織的第3代細胞表達CD13、CD44、CD73、CD90、CD166、HLA-AB,不表達CD34、CD45、HLA-DR,與骨髓來源的間充質(zhì)干細胞具有相似的細胞表面標(biāo)志。細胞在誘導(dǎo)開始時,細胞增殖速度較慢,誘導(dǎo)一周時即已停止增殖,細胞形態(tài)發(fā)生改變,細胞體積變大,部分細胞由原來的梭形逐漸變成多邊形,細胞形態(tài)不一,整個誘導(dǎo)分化過程細胞未傳代。細胞在誘導(dǎo)21d時甲苯胺藍染色可見細胞間基質(zhì)呈藍紫色。Ⅱ型膠原免疫細胞化學(xué)檢測均發(fā)現(xiàn)細胞胞漿內(nèi)有棕黃色顆粒的陽性結(jié)果。 結(jié)論 1、人胎盤來源的間充質(zhì)干細胞可成功在體外培養(yǎng)并大量擴增,生物學(xué)性狀穩(wěn)定,是組織工程優(yōu)良的種子細胞來源之一。 2、本實驗在單層培養(yǎng)條件下,成功將人胎盤間充質(zhì)干細胞誘導(dǎo)分化成軟骨細胞。經(jīng)誘導(dǎo)培養(yǎng)后形成的軟骨細胞具有軟骨細胞的形態(tài)特征,并能分泌軟骨細胞特異性Ⅱ型膠原。
[Abstract]:objective
Mesenchymal stem cells (MSCs) is a kind of pluripotent stem cell belonging to the mesoderm. It mainly exists in connective tissue and organ interstitium, and is the most abundant in bone marrow. In recent years, human bone marrow, peripheral blood and muscle have been successfully developed with the in-depth study of the biological characteristics and functions of mesenchymal stem cells. The isolation and identification of fat, umbilical blood, amniotic fluid and fetal tissues and the identification of MSCs. placenta as an important temporary organ for the exchange of oxygen and nutrients in the embryonic development of the embryo and the fetus, is composed of the fetal plexus and the mother's uterine decidua, which originate in the cell trophoblast and the mesoderm of the embryo, and from the mother's uterus decidua. The development behavior includes the more immature embryos and mature adult stem cells. The placenta completes its mission and becomes "abandoned" after the birth of the fetus. The research does not involve the ethical and moral problems. Therefore, it has become a hot spot for finding new sources of human mesenchymal stem cells and improving the effect of clinical application. In this paper, the methods of isolation and culture of Placenta-derived Mesenchymal Stem Cells (PMSCs) from human placenta and the conditions for directing differentiation of PMSCs into chondrocytes in monolayer culture are discussed.
Method
1, extraction of human placental mesenchymal stem cells
Take the placenta of the term cesarean section, peel the decidua, cut the decidua, cut the tissue under the decidua, rinse PBS for two times. Shi Wenjing set up 10min, cut the tissue block up to about 1mm~3, and put it at the bottom of the incubator evenly. The 10%DMEM medium is used to cultivate the 5%CO_2 culture box at 37 degrees C, each 3D is replaced once, and the cell line along the bottom of the incubator reaches 80% ~ 90%, according to the routine Method of passage.
2, phenotypic determination of placental cells
After taking third generations of cells and digesting 3min with 0.25% trypsin, the number of cells was adjusted to 1 x 10~6/L. After adding the 5 mu L mouse serum to close the 15min, the mouse anti human PE labeled monoclonal antibody was added to the mouse, the ice on the light reaction was 20min, and the PBS was washed 2 times, and the 5min was centrifuged at 4 1000r/min, after the supernatant was added to 200 mu L cold PBS and blended, the flow cytometer detected.
3, induced differentiation
After digestion of the third generation placental derived mesenchymal stem cells, the cell suspensions were digested. After the centrifuge was abandoned, PBS was washed 3 times, and 2 x 10~4/cm~2 density was inoculated in the six hole cell culture medium with 0.1% gelatin. After the cells were adhered to the wall, the chondrocytes were changed to the 37 C 5%CO_2 culture incubator, and each 3D was replaced once.
4, toluidine blue staining
Two groups of cells were cultured for 21d. After PBS was changed for 2 times, 4% paraformaldehyde was fixed 15 to 30min. After PBS washing for 3 times, 1% toluidine blue was stained 2 to 4h, 95% alcohol was washed for 3 times, and then observed under light microscope.
5, immunocytochemical detection of type II collagen expression
The concrete steps are operated according to the instructions of the SABC staining kit, and the culture liquid in the orifice plate is abandoned, PBS (0.02mol/LpH7.2 ~ 7.6) is washed 3 times, 4% polyformaldehyde is fixed to 30min, and 0.3%H_2O_2 is used to inactivate the endogenous enzymes. After the BSA is closed, the Rabbit anti human anti type II collagen is added to the 1:200 diluted rabbit, 4 degrees centigrade for the night, the goat is incubated with the rabbit against the rabbit mouse IgG, and the DAB chromogenic agent is coloured. The hematoxylin was observed under the light microscope after redyeing.
Result
In the tissue mass of 11d, a small number of cells were found to climb out, round or polygonal. With the number of cells increasing, the cells were vortexed, the cell body became elongated, the morphology was similar to fibroblasts, the cells were cultured about 3W, and the cells could reach 80% fusion. After the cells were adhered to the cells, the cells could grow into spindle cells again. The cells could still grow steadily to the 20 generation. Flow cells still can still grow steadily. Flow cells still can still grow. Flow cells still can still grow. Flow cells can still grow steadily. The third generation cells from human placenta tissue express CD13, CD44, CD73, CD90, CD166, HLA-AB, which do not express CD34, CD45, HLA-DR, and have similar cell surface markers with mesenchymal stem cells derived from bone marrow. Cell proliferation at the beginning of induction is slow, and the cell morphology changes at one week. The cell volume became larger and some cells were gradually transformed from the original spindle shape to polygon, and the cell morphology was different. The cells were not passable throughout the induction process. The cells were blue purple when the cells were induced by toluidine blue staining, and the positive results of the brown yellow granules in the cytoplasm of the cells were detected by the type II collagen immunocytochemical test.
conclusion
1, human placenta derived mesenchymal stem cells can be successfully cultured in vitro and amplified in large quantities, with stable biological properties.
2, in this experiment, human placental mesenchymal stem cells were successfully induced and differentiated into chondrocytes under single layer culture. The chondrocytes formed after induction were characterized by chondrocytes and could secrete the specific type II collagen of chondrocytes.
【學(xué)位授予單位】：中國醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R329

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