本文選題：骨髓間充質(zhì)干細胞 + 干細胞移植　； 參考：《安徽醫(yī)科大學》2014年碩士論文

【摘要】：目的：建立一個高效的骨髓間充質(zhì)干細胞（bone marrow mesenchymal stem cells,BMSCs）體外分離和純化的方法。 方法:取4周齡雌性ICR小鼠的脛骨和股骨進行全骨髓細胞培養(yǎng)，，采用培養(yǎng)初期換液和減少細胞傳代時胰蛋白酶消化時間（1min）富集BMSCs，通過形態(tài)學觀察和流式細胞術(shù)檢測細胞表面特異性干細胞標記物（CD29、CD44、CD45），鑒定其性質(zhì)和純度。 結(jié)果:①形態(tài)學觀察原代細胞培養(yǎng)24h可見少量散在分布、呈梭形的貼壁細胞；培養(yǎng)3d后，貼壁細胞呈集落樣生長，貼壁面積達30%，集落間可見以紅細胞為主的圓形未貼壁細胞；培養(yǎng)7d后，貼壁細胞排列緊密，呈梭形或樹突狀，細胞貼壁面積達80%。第1次傳代培養(yǎng)7d后，貼壁細胞幾乎鋪滿培養(yǎng)皿底，排列密集，貼壁面積達90%；第3次傳代培養(yǎng)5d后，梭形貼壁細胞鋪滿皿底，貼壁面積達100%，細胞飽滿、大小均一，呈旋渦狀排列。②B MSCs細胞表面標志物鑒定第3代BMSCs特異性表面標志物CD29、CD44的陽性率分別為99.24%和97.43%，而CD45呈低表達，僅為0.21%。 結(jié)論：原代培養(yǎng)的BMSCs經(jīng)傳代后生長更加迅速和穩(wěn)定；在培養(yǎng)初期通過增加換液次數(shù)、減少細胞傳代胰蛋白酶消化時間可獲取高純度的BMSCs。 目的：探討骨髓間充質(zhì)干細胞（BMSCs）移植在卵巢功能重建中的應用價值。 方法：采用環(huán)磷酰胺（CTX）120mg/kg和白消安（BUS）12mg/kg一次性腹腔注射建立卵巢早衰小鼠模型。按處理方案分為建模移植組、建模對照組和空白對照組，建模移植組于建模14d經(jīng)尾靜脈注射0.1ml綠色熒光蛋白（green fluorescentprotein, GFP）轉(zhuǎn)基因鼠BMSCs細胞懸液（5×106），建模對照組于建模14d尾靜脈注射等量生理鹽水，以均注射生理鹽水的正常健康雌性小鼠作為空白對照組。觀察小鼠的一般情況、卵巢內(nèi)卵泡數(shù)量、血清性激素FSH和E2水平，鏡下觀察建模移植組小鼠卵巢的組織學變化以及是否有綠色熒光分布。 結(jié)果：①小鼠一般情況建模組小鼠食欲明顯減退，嗜睡及活動減少，體重隨著時間推移明顯下降，建模14d體重約減輕至建模前的一半，顯著低于空白對照組；BMSCs移植后，小鼠體重平緩上升，食欲增強，活動增多。②血清FSH和E2水平測定空白對照組注射生理鹽水前后無明顯變化；建模后14d，F(xiàn)SH水平顯著高于空白對照組，E2水平則顯著降低；BMSCs移植后，F(xiàn)SH水平呈逐漸下降、E2呈逐漸上升趨勢，移植后28d FSH和E2水平與空白對照組間無顯著性差異。③卵巢組織學觀察建模14d后，總卵泡數(shù)、原始卵泡、生長卵泡和成熟卵泡均低于空白對照組，且閉鎖卵泡增多。移植后28d，各級卵泡數(shù)均顯著高于建模對照組，閉鎖卵泡減少。④熒光顯微鏡觀察BMSCs移植后28d，熒光顯微鏡下可見卵泡周圍散在分布顆粒狀綠色熒光。 結(jié)論：骨髓間充質(zhì)干細胞可定位于卵巢組織，對受損卵巢組織有修復作用，并可明顯改善卵巢的內(nèi)分泌功能。
[Abstract]:Aim: to establish an efficient method for isolation and purification of bone marrow mesenchymal stem cells (BMSCs) in vitro. Methods: the whole bone marrow cells were cultured from the tibia and femur of 4-week-old female ICR mice. BMSCs were enriched with trypsin digestion time (1 min). The cell surface specific stem cell marker, CD29, CD44and CD45, was detected by morphological observation and flow cytometry, and its properties and purity were identified. Results morphological observation showed that a small number of adherent cells were scattered in primary culture for 24 hours, and the adherent cells grew like colony after 3 days of culture, and the adhesion area was 30%, and the round unadherent cells mainly red blood cells could be seen in the colonies. After 7 days of culture, the adherent cells were closely arranged, fusiform or dendritic, and the adherent area of the cells was 80%. After the first passage for 7 days, the adherent cells were almost covered with the bottom of the petri dish, arranged densely, and the adherent area reached 90. After the third passage for 5 days, the fusiform adherent cells were covered with the bottom of the dish, and the adherent area was 100. The cells were full and uniform in size. The positive rates of CD29 and CD44 in the third generation of BMSCs were 99.24% and 97.43%, respectively, while the expression of CD45 was low (0.21%). Conclusion: the primary cultured BMSCs can grow faster and more steadily after passage, and high purity BMSCs can be obtained by increasing the number of liquid exchange at the beginning of culture and reducing the digestion time of passage trypsin. Objective: to investigate the value of bone marrow mesenchymal stem cells (BMSCs) transplantation in ovarian functional reconstruction. Methods: the mouse model of premature ovarian failure was established by intraperitoneal injection of CTX 120 mg / kg and BuUS 12 mg / kg. According to the treatment plan, it is divided into three groups: modeling transplantation group, modeling control group and blank control group. The BMSCs cell suspension of 0.1ml green fluorescent protein (GFP) transgenic mice was injected into the tail vein for 14 days in the model transplantation group and the model control group was injected with the same amount of normal saline through the tail vein on the 14th day. The normal female mice injected with normal saline were used as the blank control group. The general situation of the mice, the number of follicles in the ovary, the serum levels of FSH and E2, the histological changes of the ovaries and the distribution of green fluorescence in the transplanted mice were observed under microscope. Results in the control group, the appetite, lethargy and activity decreased significantly, and the body weight decreased significantly with time. The weight of the model group decreased to about half of that of the control group at the 14th day, which was significantly lower than that of the control group after BMSCs transplantation. The level of serum FSH and E2 in the blank control group did not change significantly before and after the injection of normal saline. On the 14th day after modeling, the level of FSH was significantly higher than that of the control group (P < 0.05), but the level of FSH decreased gradually after transplantation of BMSCs, and the level of FSH increased gradually after transplantation. There was no significant difference in the levels of FSH and E2 between the control group and the control group on the 28th day after transplantation, the total number of follicles, primordial follicles, growth follicles and mature follicles were lower than those in the control group, and the number of atretic follicles increased. On the 28th day after transplantation, the number of follicles at all levels was significantly higher than that in the model control group. At 28 days after BMSCs transplantation, the number of follicles at all levels was significantly higher than that in the model control group. Conclusion: bone marrow mesenchymal stem cells can locate in ovarian tissue, repair damaged ovarian tissue and improve endocrine function of ovary.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R711.75

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