【摘要】：目的：應(yīng)用大鼠陰莖海綿體分離、純化肌源性干細胞（muscle derived stem cells,MDSCs）,檢測干細胞標志物的表達，并將分離得到的細胞進行傳代培養(yǎng)；檢測分離細胞在特定條件下向成脂細胞及成骨細胞分化的潛能；檢測分離細胞在特定條件下向內(nèi)皮細胞分化的潛能。 方法：取2月齡SD雄性大鼠為實驗對象，用酶消化法結(jié)合percoll密度梯度離心法及改良的Preplate差速貼壁法，分離、純化大鼠陰莖海綿體肌源性干細胞。利用免疫熒光細胞化學法檢測肌源性干細胞標記物干細胞抗原1（Sca-1）和結(jié)蛋白（Desmin）在PP6細胞中的表達，并對PP6細胞進行傳代培養(yǎng)。將第3代PP6細胞在成脂及成骨誘導(dǎo)分化培養(yǎng)基中培養(yǎng)21d，同時對照組采用干細胞培養(yǎng)基培養(yǎng)；分別于誘導(dǎo)0d、7d、14d、21d通過油紅“O”染色，觀察是否有脂滴形成；通過茜素紅染色，觀察是否有成骨細胞形成的鈣結(jié)節(jié)。將第3代PP6細胞在血管內(nèi)皮細胞生長因子（vascularendothelial growth factor, VEGF）和堿性成纖維細胞生長因子（Basic fibroblast growthfactor, bFGF）共同誘導(dǎo)21d后，免疫熒光細胞化學法鑒定內(nèi)皮細胞標志物CD31及胎肝激酶-1（Flk-1）的表達；分別在誘導(dǎo)0d、5d、10d、15d、21d，通過逆轉(zhuǎn)錄-聚合酶鏈反應(yīng)（RT-PCR）檢測干細胞標志物（Oct-4）和內(nèi)皮細胞標志物CD31、內(nèi)皮型一氧化氮合酶（eNOS）、Flk-1的mRNA表達量；通過Dil-Ac-LDL攝取實驗檢測細胞吞噬低密度脂蛋白的能力；劃痕24h后觀察劃痕空白處的距離，評估細胞的遷移能力。 結(jié)果：分離、純化的PP6細胞表達肌源性干細胞標志物Sca-1和Desmin。在成脂誘導(dǎo)分化過程中，誘導(dǎo)前3d可見細胞的形狀由長梭形變?yōu)閳A形或者三角形，排列緊密，呈疊瓦樣，細胞核清晰，細胞分裂增殖明顯減慢，油紅O染色誘導(dǎo)組與對照組均未見細胞紅染；7d左右在相差顯微鏡下可觀察到胞質(zhì)內(nèi)有少量的高折光性小脂滴出現(xiàn)，大小均勻，主要集中在細胞核周圍，油紅O染成紅色；14d左右細胞內(nèi)脂滴逐漸增多并融合成大脂滴，細胞核被擠到一邊或者消失；誘導(dǎo)21d細胞分化達高峰，，細胞體積達到最大，胞質(zhì)內(nèi)脂滴數(shù)量最多，繼續(xù)誘導(dǎo)，細胞內(nèi)未見空泡增多。而對照組細胞形態(tài)未發(fā)生變化，且油紅O染色陰性。在成骨誘導(dǎo)分化過程中，誘導(dǎo)前7d可見細胞的形狀由長梭形變?yōu)閳A形或者三角形，排列緊密，細胞核清晰，細胞分裂增殖明顯減慢，茜素紅染色兩組均未見鈣結(jié)節(jié)形成；誘導(dǎo)14d后，細胞出現(xiàn)層疊、聚集生長態(tài)勢，融合的細胞呈現(xiàn)“疊瓦樣”改變，細胞體積變大、形態(tài)呈多邊形，核大，核仁清晰，細胞內(nèi)顆粒增多，細胞外有少量基質(zhì)分泌，茜素紅染色可見散在分布的鈣結(jié)節(jié)形成；誘導(dǎo)21d時鏡下可見細胞內(nèi)顆粒增多，細胞外基質(zhì)分泌旺盛，茜素紅染色顯示細胞間紅色礦化基質(zhì)沉淀，鈣結(jié)節(jié)形成。在內(nèi)皮細胞誘導(dǎo)分化過程中，PP6誘導(dǎo)21d后表達內(nèi)皮細胞標志物（CD31和Flk-1）；誘導(dǎo)21d后與誘導(dǎo)0d比較，干細胞標志物Oct-4的mRNA表達量下降（下降倍數(shù)為0.445±0.025），而內(nèi)皮細胞標志物mRNA表達量升高（CD31、eNOS、Flk-1升高倍數(shù)分別為2.541±0.146、2.364±0.175、2.538±0.100），差異均有統(tǒng)計學意義（P0.05）；Dil-Ac-LDL攝取實驗提示誘導(dǎo)后的細胞具有吞噬低密度脂蛋白的能力；劃痕實驗觀察到誘導(dǎo)組空白處距離為282.01±3.84，未誘導(dǎo)組空白處距離為450.35±1.86，差異有統(tǒng)計學意義（P0.05）。 結(jié)論：大鼠陰莖海綿體中分離純化的細胞表達肌源性干細胞標記物，并且在特定條件下可分化為成脂細胞、成骨細胞及內(nèi)皮細胞，進一步證實了陰莖海綿體中存在肌源性干細胞。
[Abstract]:Objective: To detect the expression of stem cell marker in rat corpus cavernosum, purify muscle source stem cells (MDSCs), to detect the expression of stem cell markers, and to detect the potential of isolated cells to differentiate into adipocytes and osteoblasts under specific conditions; Detection of the potential of isolated cells to differentiate into endothelial cells under specific conditions. Methods: Two-month-old SD male rats were taken as experimental subjects, and percoll density gradient centrifugation and modified Preplate differential attachment method were combined with enzyme digestion to separate and purify the muscular source of corpus cavernosum of rats. The expression of Sa-1 and Desmin in PP6 cells was detected by immunofluorescence cytochemical method, and the P6 cells were passaged. culturing the 3rd generation PP6 cells in lipoid and bone inducing differentiation medium, and culturing in the control group by using stem cell culture medium; respectively inducing 0d, 7d, 14d, 21d; over-oiled red "O" staining to see if lipid droplets were formed; by staining with Congo red, observe whether osteoblasts were formed or not. The expression of the endothelial cell marker CD31 and fetal liver kinase-1 (Flk-1) was identified by immunofluorescence cytochemical method after 21days induction by vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). 5d, 10d, 15d, 21d, detecting stem cell markers (Oct-4) and endothelial cell markers CD31, endothelial nitric oxide synthase (VEGF) and Flk-1 mRNA expression by reverse transcription-polymerase chain reaction (RT-PCR); Ability to evaluate cell migration by observing the distance in the blank space after scratch 24h Ability. Results: Isolated, purified PP6 cells express muscle-derived stem cell markers Sca-1 and D In the process of lipoid induction and differentiation, the shape of the visible cells induced by the induction of lipoid induction was circular or triangular in shape of long shuttle, closely aligned, stacked tiles, clear cell nucleus, obvious decrease in cell division and proliferation, and neither the oil red O staining induction group nor the control group It can be seen that a small amount of high-refractive small lipid droplets appear in the cytoplasm and the size is even, mainly in the periphery of the nucleus, the oil red O is dyed red, and the fat drops in the left and right cells are gradually increased and fused into big fat drops, and the nuclei are squeezed to one. The differentiation of 21d cells reached the peak, the volume of cells reached the maximum, the number of intracellular lipid droplets was the most, continued to induce, and the cells were not In the control group, the morphology of the cells did not change and the oil was red. O staining was negative. In the process of bone induction and differentiation, the shape of the visible cells was circular or triangular in shape of long shuttle, the arrangement was tight, the cell nucleus was clear, cell division and proliferation were obviously slowed down, and no calcium nodules were found in the two groups. The cells were laminated, aggregated and grown, the fused cells showed 鈥渟tacked tile鈥

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