本文選題：富血小板血漿　切入點(diǎn)：骨髓基質(zhì)細(xì)胞　出處：《青島大學(xué)》2005年碩士論文

【摘要】：目的:體外觀察不同濃度富血小板血漿(PRP)對(duì)自體骨髓基質(zhì)細(xì)胞堿性磷酸酶活性和mRNA表達(dá)的影響。對(duì)PRP誘導(dǎo)骨髓基質(zhì)細(xì)胞向成骨細(xì)胞分化的生物學(xué)效應(yīng)進(jìn)行初步評(píng)價(jià)。 方法:抽取狗靜脈血,采用改進(jìn)Appel方法制備PPP和不同血小板濃度的PRP。取狗骨髓,于含15%胎牛血清的D-MEM-F12培養(yǎng)液中培養(yǎng),經(jīng)傳代培養(yǎng)篩選,獲得足夠數(shù)量穩(wěn)定的骨髓基質(zhì)細(xì)胞(BMSCs)。實(shí)驗(yàn)分5組,分別加入誘導(dǎo)物(A組PPP+BMSCs,B組PRP+BMSCs,C組4倍PRP+BMSCs,D組rhBMP+BMSCs,E組為空白對(duì)照組),分別在第3、6、9、12天用酶動(dòng)力學(xué)法檢測(cè)堿性磷酸酶活性,用RT-PCR法在第6天檢測(cè)堿性磷酸酶mRNA的表達(dá),Von kossa染色鑒定成骨細(xì)胞。 結(jié)果:(1)原代培養(yǎng)的骨髓基質(zhì)細(xì)胞24h開始貼壁,12d后細(xì)胞融匯成單層,細(xì)胞多呈成纖維梭形細(xì)胞形態(tài),經(jīng)誘導(dǎo)液傳代培養(yǎng)后,細(xì)胞形態(tài)漸變?yōu)榉叫�、多角形�?2)酶動(dòng)力學(xué)法測(cè)定各實(shí)驗(yàn)組細(xì)胞第3、6、9、12天檢測(cè)堿性磷酸酶活性,經(jīng)過配對(duì)t檢驗(yàn),各組第6、9、12天的ALP活性水平均較第3天明顯升高,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。(3)用RT-PCR法檢測(cè)第6天堿性磷酸酶mRNA的表達(dá),結(jié)果顯示PRP低濃度組ALPmRNA表達(dá)明顯高于對(duì)照組。(4)Von kossa染色鑒定成骨細(xì)胞:細(xì)胞密集區(qū)出現(xiàn)大面積黑染區(qū)域,即鈣結(jié)節(jié)。 結(jié)論:一定濃度的PRP能誘導(dǎo)骨髓基質(zhì)細(xì)胞堿性磷酸酶mRNA的表達(dá)增加,并提高細(xì)胞堿性磷酸酶的活性,PRP能有效的促使BMSCs向前期成骨細(xì)胞分化。骨組織學(xué)觀察證實(shí)PRP能誘導(dǎo)BMSCs向成骨細(xì)胞分化。
[Abstract]:Aim: to investigate the effects of different concentrations of platelet-rich plasma on alkaline phosphatase (ALP) activity and mRNA expression in autologous bone marrow stromal cells (BMSCs).The biological effects of PRP induced bone marrow stromal cell differentiation into osteoblasts were preliminarily evaluated.Methods: PPP and different platelet concentrations were prepared by modified Appel method.The bone marrow of dog was cultured in D-MEM-F12 medium containing 15% fetal bovine serum, and a sufficient number of stable bone marrow stromal cells (BMSCs) were obtained by subculture and screening.The experiment was divided into five groups. The activity of alkaline phosphatase was detected by enzyme kinetic method on the 12th day of the 3rd day after the addition of inducer A, PPP BMSCs, B, PRP BMSCs, C, 4 times PRP BMSCs, D, rhBMP, BMSCs, and E, as blank control group.The expression of alkaline phosphatase (mRNA) in osteoblasts was detected by RT-PCR method and Von kossa staining was used to identify osteoblasts.Results the primary cultured bone marrow stromal cells were fused into a monolayer after 12 days of adherent adhesion. The cells were mostly fibroblast-like cells. After the passage of the induction medium, the cell morphology gradually changed to square.The activity of alkaline phosphatase (ALP) was measured by polygonal kinetic assay on the 12th day of the 3rd day, and the activity of ALP was significantly higher than that of the 3rd day after paired t test.The expression of alkaline phosphatase mRNA was detected by RT-PCR on the 6th day. The results showed that the expression of ALPmRNA in the low PRP group was significantly higher than that in the control group. Von kossa staining was used to identify the osteoblasts.Calcium nodules.Conclusion: PRP can induce the expression of alkaline phosphatase mRNA in bone marrow stromal cells and increase the activity of alkaline phosphatase in bone marrow stromal cells. It can effectively induce the differentiation of BMSCs into osteoblasts.Bone histological observation showed that PRP could induce BMSCs to differentiate into osteoblasts.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R329.2

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相關(guān)期刊論文 前2條

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本文編號(hào)：1709281