冲击波与地塞米松诱导自体血清培养的hMSCs成骨分化的实验研究

发布时间：2018-03-20 00:23

本文选题：体外冲击波疗法　切入点：骨髓间充质干细胞　出处：《河北医科大学》2010年硕士论文　论文类型：学位论文

【摘要】： 目的:人骨髓间充质干细胞(human mesenchymal stem cells,hMSCs)是从骨髓中分离得到的一种多潜能干细胞,具有自我更新和多向分化潜能,已证实这类细胞在合适的体内外特定条件下可向成骨、软骨、神经、肌肉、皮肤和肝等多种类型的成熟细胞分化,因而是组织工程领域中的理想的种子细胞。但由于骨髓中hMSCs的数量有限,在进行体内移植前必须进行体外扩增。目前主要应用胎牛血清(fetal bovine serum,FBS)进行hMSCs的体外培养,而胎牛血清本身可能传染病毒、带来异种免疫原,且存在排斥反应的问题,这极大地阻碍了hMSCs在临床上的应用,而自体血清(autologous serum,AS)可避免上述问题。而对hMSCs进行成骨诱导主要应用传统的地塞米松,但地塞米松浓度的差异对hMSCs的影响很大,浓度稍微增大,对细胞增殖有明显抑制作用,并有向脂肪细胞诱导分化的趋势。体外冲击波疗法(extracorporeal shock waves therapy,ESWT )因其能够显著促进成骨细胞增殖,近年来利用其治疗股骨头缺血性坏死、骨不连及骨折延迟愈合等取得了良好的疗效。我们利用ESWT与地塞米松比较对AS培养的hMSCs进行成骨分化。本研究国内外均未见报道。本研究利用ESWT与地塞米松比较对AS培养的hMSCs进行成骨分化,为临床治疗骨病探索一种新的治疗方法,从而指导临床治疗。方法: 1抽取分离hMSCs及获取AS:选择10名志愿者(排除代谢性疾病),每名志愿者抽取骨髓60ml,用梯度离心法分离出hMSCs。抽取外周静脉血100ml,然后分离出AS(约55ml)。 2观察AS对hMSCs增殖的影响:把每名志愿者的骨髓分成两组,分别用10%AS和10%FBS对hMSCs进行体外培养,通过相差倒置显微镜观察细胞形态、检测24小时细胞贴壁率、细胞倍增时间、CCK-8(cell counting kit-8)测细胞增殖曲线、检测细胞表面标记物、细胞周期及免疫细胞化学染色检测比较两组hMSCs的增殖状况。 3比较ESWT与地塞米松对AS培养的hMSCs成骨分化的影响:把AS培养的hMSCs分成两组,两组分别用ESWT(0.36mj/mm2/100次)和传统的诱导成骨培养基(10nM地塞米松、50uM抗坏血酸、10mMβ-甘油磷酸钠)分别对hMSCs进行成骨诱导,对诱导好的细胞进行ALP定量检测、ALP染色、茜素红染色及RT-PCR测成骨基因(碱性磷酸酶、骨钙素、骨桥蛋白、骨结合素、Ⅰ型胶原)表达比较两组成骨分化情况。对所有数据x±s表示统计学分析,设定P0.05为有统计学意义。结果: 1 hMSCs显微镜下细胞形态学观察:AS和FBS培养的细胞在同一代次上形态无明显差异,具有典型的hMSCs形态,随着代次的增加,部分细胞逐渐变扁,贴壁性减退,出现一些漂浮的细胞;部分分泌强的细胞分泌物增多,细胞增殖速度明显下降,这种现象在7代以后尤为明显。根据P3细胞计数结果计算24h贴附率,两组组分别为:(94.3±1.7)%、(93.0±1.6)%,(P0.05),无显著差异。根据P3细胞计数AS组细胞倍增时间(平均53小时,41-54h),FBS组(平均84小时,76-89h),P0.01,有显著性差异,AS组的细胞倍增时间明显短于FBS组。CCK-8检测根据每日检测结果绘制细胞增殖曲线显示两组间差异非常显著(P0.01),AS培养的hMSCs增殖能力提高,峰值增高。AS在细胞增殖上明显优于FBS。 2细胞表面分子测定和免疫细胞化学染色检测:流式细胞仪检测结果显示AS和FBS培养的细胞表面抗原CD44的表达阳性率分别为99.96%、99.30%,符合hMSCs的细胞表面特征,两组没有明显差异。免疫荧光染色显示两组细胞CD44、CD105均呈阳性表达,绿色、红色荧光遍及胞质。 3 hMSCs细胞周期测定:结果显示hMSCs具有干细胞的典型周期特性,大部分细胞处于G0 /G1期,只有少数进入S期。随代次增加,处于G0 /G1期的细胞的比例逐渐减少,S期细胞比例逐渐增多,与形态及生长特性的变化规律相一致,反映了细胞衰老的进程,两组没有明显差异。 4 ALP定量测定和ALP染色:结果显示在各时间点ESWT组ALP活性明显高于地塞米松诱导组,两组都在第四天开始持续增加,在第14天达到峰值,继而下降。两组有明显差异(P0.05)。两组同样分别成骨诱导培养14天后,弃培养基,用PBS洗剂2次,用95%酒精固定10分钟。按ALP试剂盒说明书进行染色。结果显示ESWT组ALP染色强度明显高于地塞米松诱导组。 5茜素红法钙化结节染色:两组同样分别诱导28天后进行茜素红染色,两组均可见矿化结节。100倍镜下随机选取3个视野进行计数,结果ESWT组矿化结节数为7.5±1.2,地塞米松诱导组矿化节数为5.0±0.8,ESWT组钙结节数量明显多于地塞米松诱导组。 6 RT-PCR检测成骨基因表达:两组同样分别成骨诱导培养28天后检测:碱性磷酸酶(alkaline phosphatase,ALP)、骨钙素(osteocalcin ,OC),骨桥蛋白(osteopontin ,OP),骨结合素(osteonectin ,ON),Ⅰ型胶原( collagenⅠ, ColⅠ), GAPDH ( glyceraldehyde-3-phosphate dehydrogenase )表达情况。成骨诱导28天后,冲击波干预组中的ALP、ON、OP、OC、ColⅠ的基因表达明显高于地塞米松诱导组。结论: 1、自体血清与胎牛血清相比,对人骨髓间充质干细胞更具有良好的促增殖作用。我们选择AS培养的hMSCs进行成骨诱导。 2、冲击波作用于人骨髓间充质干细胞的成骨效应明显优于地塞米松,因而是一种更好的诱导成骨分化的刺激方式。
[Abstract]:Objective: human bone marrow mesenchymal stem cells (human mesenchymal stem cells, hMSCs) were isolated from bone marrow of a pluripotent stem cells with self-renewal and differentiation potential, has confirmed that these cells from bone, in appropriate in vivo specific conditions of cartilage, nerve, muscle, mature cell differentiation of skin and liver and other types, so it is the seed cells for tissue engineering in the field of ideal. But due to the limited number of bone marrow transplantation in vivo in hMSCs, must be carried out in vitro. The main application of fetal bovine serum (fetal bovine serum, FBS hMSCs) were cultured in vitro, and fetal bovine serum may the infectious virus, bring heteroimmune, and rejection problem, which greatly hinder the application of hMSCs in clinic, and autologous serum (autologous serum, AS) can avoid the problem of hMSCs into. The main application of the traditional bone induced by dexamethasone, but a great impact on the difference of concentration of dexamethasone hMSCs, concentration increased slightly, has obvious inhibitory effects on cell proliferation and differentiation into fat cells. The trend of extracorporeal shock wave therapy (extracorporeal shock waves therapy, ESWT) because it can significantly promote the proliferation of osteoblasts, in recent years to use the treatment of ischemic necrosis of femoral head, bone nonunion and delayed fracture healing and achieved good results. We use ESWT and dexamethasone compared to AS cultured hMSCs of osteogenic differentiation. The research at home and abroad are reported. This study compared to AS cultured hMSCs osteogenic differentiation by ESWT and dexamethasone for the clinical treatment of bone disease, to explore a new treatment method, so as to guide the clinical treatment.
Method:
1 extract and separate hMSCs and get AS: to select 10 volunteers (exclude metabolic diseases). Each volunteer extracts 60ml from bone marrow, and extracts hMSCs. from peripheral vein blood by gradient centrifugation, then separates AS (about 55ml).
To observe the effects of 2 AS on the proliferation of hMSCs: each bone marrow volunteers were divided into two groups, respectively, the hMSCs were cultured in vitro with 10%AS and 10%FBS, the cell morphology was observed by inverted microscope, detected 24 hours cell adhesion rate, cell doubling time, CCK-8 (cell counting kit-8) to measure the cell proliferation curve, detection of cell surface markers, cell cycle and immunocytochemical staining were performed to detect the proliferation of hMSCs were compared between the two groups.
3 Comparison of ESWT and dexamethasone on osteoblastic differentiation of cultured AS hMSCs AS in the cultured hMSCs were divided into two groups, two groups were treated with ESWT (0.36mj/mm2/100) and traditional osteogenic medium (10nM 50uM 10mM, dexamethasone, ascorbic acid, sodium glycerophosphate) of hMSCs osteogenic differentiation. ALP quantitative detection of the induced cells by ALP staining, alizarin red staining and RT-PCR osteogenic genes (alkaline phosphatase, osteocalcin, osteopontin, osteonectin, type I collagen) expression of two bone differentiation. Statistical analysis of all data set of X + s, P0.05 had statistical significance.
Result:
Observation of 1 hMSCs cell morphology under microscope: there was no significant difference in AS and FBS cells cultured in the same generation time on morphology, with typical morphology of hMSCs, with the increase of generation time, part of the cells became flat, adherent loss of some floating cells; some strong secretion cell secretion and cell proliferation the speed decreased, this phenomenon is particularly evident in the 7 generation. According to the calculation results of 24h P3 cell attachment rate, the two groups were: (94.3 + 1.7)% and (93 + 1.6)%, (P0.05), no significant difference. According to the P3 cell count in AS group cell doubling time (an average of 53 h, 41-54h), group FBS (average 84 hours, 76-89h, P0.01), there was significant difference between AS group, the cell doubling time was significantly shorter in the FBS group according to the daily.CCK-8 detection test results draw cell growth curve showed that the difference between the two groups was very significant (P0.01, AS) to improve the ability of proliferation of cultured hMSCs, The increase of peak value of.AS in cell proliferation is obviously better than that of FBS.
2 cell surface molecule assay and immunocytochemical staining. Flow cytometry results showed that the positive expression rate of CD44 cell surface antigen AS and FBS culture were 99.96%, 99.30%, with cell surface characteristics of hMSCs, the two groups had no significant difference. Immunofluorescence staining showed that the two groups of cells were CD44, CD105 the positive expression of green and red fluorescence in the cytoplasm.
3 hMSCs cell cycle analysis: the results showed that hMSCs has the characteristics of a typical cycle of stem cells, most cells in G0 /G1 phase into S phase. With only a few generations increase in G0 /G1 phase cells proportion gradually decreased, the proportion of cells in S phase increased gradually, the change trend is consistent with the morphology and growth characteristics. Reflects the cell aging process, the two groups had no obvious difference.
Determination of 4 quantitative ALP and ALP staining: the results showed that the ESWT group at different time point of ALP was significantly higher than that induced by dexamethasone group, two groups in the fourth day continued to increase, and reached the peak on the fourteenth day, then decreased. The two groups were significantly different (P0.05). The two groups were the same bone after 14 days of induction, abandoned medium with PBS Lotion 2 times with 95% alcohol for 10 minutes. According to the fixed ALP kit were used. The results show that the ESWT group ALP staining intensity was significantly higher than that induced by dexamethasone group.
5 calcified nodules were stained by alizarin red method: two group respectively after 28 days of induction by alizarin red staining, two were found in mineralized nodules.100 times randomly select 3 scopes were counted. Results in the ESWT group, the number of calcified nodules was 7.5 + 1.2, DEX group was 5 + 0.8 mineralization section number, the number of ESWT group of calcium nodules induced by dexamethasone was significantly more than group.
6 RT-PCR detection of osteogenic gene expression: the two group also were detected in 28 days of osteogenic induction, alkaline phosphatase (alkaline phosphatase, ALP), osteocalcin (osteocalcin, OC), osteopontin (osteopontin, OP) and osteonectin (osteonectin, ON), collagen type I (collagen I, Col I, GAPDH) (glyceraldehyde-3-phosphate dehydrogenase). The expression of osteogenic induction for 28 days, the shock wave in the intervention group ALP, ON, OP, OC, Col I gene expression was significantly higher than that induced by dexamethasone group.
Conclusion:
1, the autologous serum has a better proliferation promoting effect on human bone marrow mesenchymal stem cells compared with the fetal bovine serum. We selected hMSCs for AS culture to induce osteogenesis.
2, the osteogenesis effect of shock wave on human bone marrow mesenchymal stem cells is obviously better than that of dexamethasone, thus it is a better way to induce osteogenic differentiation.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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