本文選題：體外沖擊波療法　切入點：骨髓間充質(zhì)干細胞　出處：《河北醫(yī)科大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的:人骨髓間充質(zhì)干細胞(human mesenchymal stem cells,hMSCs)是從骨髓中分離得到的一種多潛能干細胞,具有自我更新和多向分化潛能,已證實這類細胞在合適的體內(nèi)外特定條件下可向成骨、軟骨、神經(jīng)、肌肉、皮膚和肝等多種類型的成熟細胞分化,因而是組織工程領(lǐng)域中的理想的種子細胞。但由于骨髓中hMSCs的數(shù)量有限,在進行體內(nèi)移植前必須進行體外擴增。目前主要應(yīng)用胎牛血清(fetal bovine serum,FBS)進行hMSCs的體外培養(yǎng),而胎牛血清本身可能傳染病毒、帶來異種免疫原,且存在排斥反應(yīng)的問題,這極大地阻礙了hMSCs在臨床上的應(yīng)用,而自體血清(autologous serum,AS)可避免上述問題。而對hMSCs進行成骨誘導(dǎo)主要應(yīng)用傳統(tǒng)的地塞米松,但地塞米松濃度的差異對hMSCs的影響很大,濃度稍微增大,對細胞增殖有明顯抑制作用,并有向脂肪細胞誘導(dǎo)分化的趨勢。體外沖擊波療法(extracorporeal shock waves therapy,ESWT )因其能夠顯著促進成骨細胞增殖,近年來利用其治療股骨頭缺血性壞死、骨不連及骨折延遲愈合等取得了良好的療效。我們利用ESWT與地塞米松比較對AS培養(yǎng)的hMSCs進行成骨分化。本研究國內(nèi)外均未見報道。本研究利用ESWT與地塞米松比較對AS培養(yǎng)的hMSCs進行成骨分化,為臨床治療骨病探索一種新的治療方法,從而指導(dǎo)臨床治療。 方法: 1抽取分離hMSCs及獲取AS:選擇10名志愿者(排除代謝性疾病),每名志愿者抽取骨髓60ml,用梯度離心法分離出hMSCs。抽取外周靜脈血100ml,然后分離出AS(約55ml)。 2觀察AS對hMSCs增殖的影響:把每名志愿者的骨髓分成兩組,分別用10%AS和10%FBS對hMSCs進行體外培養(yǎng),通過相差倒置顯微鏡觀察細胞形態(tài)、檢測24小時細胞貼壁率、細胞倍增時間、CCK-8(cell counting kit-8)測細胞增殖曲線、檢測細胞表面標(biāo)記物、細胞周期及免疫細胞化學(xué)染色檢測比較兩組hMSCs的增殖狀況。 3比較ESWT與地塞米松對AS培養(yǎng)的hMSCs成骨分化的影響:把AS培養(yǎng)的hMSCs分成兩組,兩組分別用ESWT(0.36mj/mm2/100次)和傳統(tǒng)的誘導(dǎo)成骨培養(yǎng)基(10nM地塞米松、50uM抗壞血酸、10mMβ-甘油磷酸鈉)分別對hMSCs進行成骨誘導(dǎo),對誘導(dǎo)好的細胞進行ALP定量檢測、ALP染色、茜素紅染色及RT-PCR測成骨基因(堿性磷酸酶、骨鈣素、骨橋蛋白、骨結(jié)合素、Ⅰ型膠原)表達比較兩組成骨分化情況。對所有數(shù)據(jù)x±s表示統(tǒng)計學(xué)分析,設(shè)定P0.05為有統(tǒng)計學(xué)意義。 結(jié)果: 1 hMSCs顯微鏡下細胞形態(tài)學(xué)觀察:AS和FBS培養(yǎng)的細胞在同一代次上形態(tài)無明顯差異,具有典型的hMSCs形態(tài),隨著代次的增加,部分細胞逐漸變扁,貼壁性減退,出現(xiàn)一些漂浮的細胞;部分分泌強的細胞分泌物增多,細胞增殖速度明顯下降,這種現(xiàn)象在7代以后尤為明顯。根據(jù)P3細胞計數(shù)結(jié)果計算24h貼附率,兩組組分別為:(94.3±1.7)%、(93.0±1.6)%,(P0.05),無顯著差異。根據(jù)P3細胞計數(shù)AS組細胞倍增時間(平均53小時,41-54h),FBS組(平均84小時,76-89h),P0.01,有顯著性差異,AS組的細胞倍增時間明顯短于FBS組。CCK-8檢測根據(jù)每日檢測結(jié)果繪制細胞增殖曲線顯示兩組間差異非常顯著(P0.01),AS培養(yǎng)的hMSCs增殖能力提高,峰值增高。AS在細胞增殖上明顯優(yōu)于FBS。 2細胞表面分子測定和免疫細胞化學(xué)染色檢測:流式細胞儀檢測結(jié)果顯示AS和FBS培養(yǎng)的細胞表面抗原CD44的表達陽性率分別為99.96%、99.30%,符合hMSCs的細胞表面特征,兩組沒有明顯差異。免疫熒光染色顯示兩組細胞CD44、CD105均呈陽性表達,綠色、紅色熒光遍及胞質(zhì)。 3 hMSCs細胞周期測定:結(jié)果顯示hMSCs具有干細胞的典型周期特性,大部分細胞處于G0 /G1期,只有少數(shù)進入S期。隨代次增加,處于G0 /G1期的細胞的比例逐漸減少,S期細胞比例逐漸增多,與形態(tài)及生長特性的變化規(guī)律相一致,反映了細胞衰老的進程,兩組沒有明顯差異。 4 ALP定量測定和ALP染色:結(jié)果顯示在各時間點ESWT組ALP活性明顯高于地塞米松誘導(dǎo)組,兩組都在第四天開始持續(xù)增加,在第14天達到峰值,繼而下降。兩組有明顯差異(P0.05)。兩組同樣分別成骨誘導(dǎo)培養(yǎng)14天后,棄培養(yǎng)基,用PBS洗劑2次,用95%酒精固定10分鐘。按ALP試劑盒說明書進行染色。結(jié)果顯示ESWT組ALP染色強度明顯高于地塞米松誘導(dǎo)組。 5茜素紅法鈣化結(jié)節(jié)染色:兩組同樣分別誘導(dǎo)28天后進行茜素紅染色,兩組均可見礦化結(jié)節(jié)。100倍鏡下隨機選取3個視野進行計數(shù),結(jié)果ESWT組礦化結(jié)節(jié)數(shù)為7.5±1.2,地塞米松誘導(dǎo)組礦化節(jié)數(shù)為5.0±0.8,ESWT組鈣結(jié)節(jié)數(shù)量明顯多于地塞米松誘導(dǎo)組。 6 RT-PCR檢測成骨基因表達:兩組同樣分別成骨誘導(dǎo)培養(yǎng)28天后檢測:堿性磷酸酶(alkaline phosphatase,ALP)、骨鈣素(osteocalcin ,OC),骨橋蛋白(osteopontin ,OP),骨結(jié)合素(osteonectin ,ON),Ⅰ型膠原( collagenⅠ, ColⅠ), GAPDH ( glyceraldehyde-3-phosphate dehydrogenase )表達情況。成骨誘導(dǎo)28天后,沖擊波干預(yù)組中的ALP、ON、OP、OC、ColⅠ的基因表達明顯高于地塞米松誘導(dǎo)組。 結(jié)論: 1、自體血清與胎牛血清相比,對人骨髓間充質(zhì)干細胞更具有良好的促增殖作用。我們選擇AS培養(yǎng)的hMSCs進行成骨誘導(dǎo)。 2、沖擊波作用于人骨髓間充質(zhì)干細胞的成骨效應(yīng)明顯優(yōu)于地塞米松,因而是一種更好的誘導(dǎo)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.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R329

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