本文選題：骨髓基質(zhì)干細(xì)胞 + 生物蛋白膠　； 參考：《昆明醫(yī)學(xué)院》2011年碩士論文

【摘要】：·目的 研究5-溴脫氧尿嘧啶核苷(BrdU)標(biāo)記骨髓基質(zhì)干細(xì)胞(BMSCs)的效果,確定其體外最佳標(biāo)記濃度和最佳標(biāo)記時(shí)間；通過(guò)生物蛋白膠(FS)與標(biāo)記有BrdU的BMSCs體外成骨與共同培養(yǎng),觀察BrdU和FS對(duì)BMSCs生長(zhǎng)、增殖及成骨分化的影響,探討生物蛋白膠作為細(xì)胞載體支架的可行性,為Brdu標(biāo)記后的BMSCs/FS工程化組織應(yīng)用于體內(nèi)試驗(yàn)提供實(shí)驗(yàn)依據(jù)。 方法 1.體外分離培養(yǎng)BMSCs并檢測(cè)BrdU標(biāo)記的最佳濃度及最佳時(shí)間：取2月齡健康新西蘭大白兔,無(wú)菌狀態(tài)下取出雙側(cè)股骨,采用全骨髓培養(yǎng)法,運(yùn)用流式細(xì)胞儀,通過(guò)CD34、CD44、CD90等細(xì)胞表型鑒定,證實(shí)為純度高的BMSCs后,取P3代BMSCs,以1×105/ml細(xì)胞密度接種于16塊6孔培養(yǎng)板內(nèi)(每塊培養(yǎng)板內(nèi)預(yù)先放置蓋玻片行細(xì)胞爬片),每4塊培養(yǎng)板為一組,共四組(A、B、C、D組)：分別加入含BrdU終濃度為5umol/L、10umol/L、15umol/L、20umol/L的L-DMEM完全培養(yǎng)基避光培養(yǎng)。在培養(yǎng)后24h、48h、72h、96h同一時(shí)間段,取每一組的一塊培養(yǎng)板內(nèi)的細(xì)胞爬片行細(xì)胞免疫組化染色,計(jì)算細(xì)胞標(biāo)記率,比較不同濃度及不同時(shí)間的BrdU陽(yáng)性細(xì)胞標(biāo)記率,確定BrdU標(biāo)記的最佳濃度和最佳時(shí)間。 2.生物蛋白膠鋪于培養(yǎng)板底后植入標(biāo)記有BrdU的BMSCs體外成骨培養(yǎng)：采用上述研究的最佳BrdU標(biāo)記濃度進(jìn)行骨髓基質(zhì)干細(xì)胞(Bone Mesenchymal stem cells BMSCs)標(biāo)記,實(shí)驗(yàn)分三組：A組(對(duì)照組)：未標(biāo)記的P3代BMSCs加入成骨誘導(dǎo)培養(yǎng)基培養(yǎng)；B組：(BrdU組)：標(biāo)記有BrdU的P3代BMSCs加入成骨誘導(dǎo)培養(yǎng)基避光培養(yǎng)；C組：(實(shí)驗(yàn)組)：將生物蛋白膠以薄層平鋪于培養(yǎng)板底,再接種已標(biāo)記有BrdU的BMSCs,加入成骨誘導(dǎo)培養(yǎng)基避光培養(yǎng)。按照后面檢測(cè)目的將三組細(xì)胞植入24、96孔培養(yǎng)板內(nèi)培養(yǎng),每次隨機(jī)選取培養(yǎng)板內(nèi)6孔標(biāo)本作為檢測(cè)個(gè)數(shù)。在培養(yǎng)2、4、6、8、10、12h時(shí)行細(xì)胞貼壁率檢測(cè),成骨誘導(dǎo)1、2、4、7、14、21天后,分別進(jìn)行光鏡下大體觀察、生長(zhǎng)動(dòng)力學(xué)(MTT)檢測(cè)、臺(tái)盼藍(lán)染色、GENMED細(xì)胞堿性磷酸酶(ALP)活性染色和GENMED細(xì)胞馮庫(kù)薩(Von-Kossa)改良法染色,同時(shí)行堿性磷酸酶定量和游離鈣定量檢測(cè)(甲基百里香酚藍(lán)比色法)。 3.標(biāo)記有BrdU的BMSCs直接融入生物蛋白膠中共同培養(yǎng)：選取標(biāo)記有BrdU的P3代BMSCs,消化離心后行細(xì)胞計(jì)數(shù),約1×107個(gè)BMSCs混入纖維蛋白原溶液管內(nèi)(約5m1),與等量的凝血酶管內(nèi)液體混合后,即形成標(biāo)記有BrdU的BMSCs/FS復(fù)合物,鋪于培養(yǎng)板底,加入L-DMEM完全培養(yǎng)基培養(yǎng),為實(shí)驗(yàn)組；對(duì)照組為未標(biāo)記有BrdU勺P3代BMSCs植入培養(yǎng)板中加入L-DMEM完全培養(yǎng)基培養(yǎng)。分別在1、3、7、14、21天光鏡下觀察實(shí)驗(yàn)組中BMSCs在生物蛋白膠中生長(zhǎng)、增殖等情況；同時(shí)行FDA—PI雙色熒光法檢測(cè)活細(xì)胞率,判斷BMSCs在生物蛋白膠中存活率；最后檢測(cè)兩組BMSCs向成骨細(xì)胞分化的指標(biāo)：堿性磷酸酶(ALP)活性。以此研究BMSCs在同時(shí)存在BrdU和生物蛋白膠條件下的生長(zhǎng)、增殖與分化情況,判斷體外生物蛋白膠是否對(duì)標(biāo)記BrdU勺骨髓基質(zhì)干細(xì)胞生長(zhǎng)、增殖存在影響以及生物蛋白膠體外降解狀況。 以上實(shí)驗(yàn)數(shù)據(jù)采用SPSS17.0統(tǒng)計(jì)軟件進(jìn)行統(tǒng)計(jì)分析,P0.05有統(tǒng)計(jì)學(xué)意義。結(jié)果 1.體外分離培養(yǎng)BMSCs并檢測(cè)BrdU標(biāo)記的最佳濃度及最佳時(shí)間：P3代BMSCs通過(guò)流式細(xì)胞儀檢測(cè)結(jié)果顯示,骨髓基質(zhì)干細(xì)胞純度達(dá)90%以上。BrdU對(duì)細(xì)胞核標(biāo)記肯定,可見(jiàn)細(xì)胞核標(biāo)記后呈棕紅色。在標(biāo)記濃度方面,同一時(shí)間點(diǎn)四組不同濃度標(biāo)記率檢測(cè),10umol/L、15umol/L、20umol/L濃度標(biāo)記率較5umol/L濃度標(biāo)記率,具有顯著性差異(P0.05); 10umol/L、15umol/L、20umol/L濃度標(biāo)記率相比較,未見(jiàn)顯著性差異(P0.05)。在標(biāo)記時(shí)間方面,四組中,同組內(nèi)48h、72h、96h標(biāo)記率與24h標(biāo)記率相比,具有顯著性差異(P0.05)：同組中48h、72h、96h標(biāo)記率相比,P0.05,無(wú)統(tǒng)計(jì)學(xué)意義。故說(shuō)明在10umol/L濃度下及標(biāo)記48h是最佳標(biāo)記濃度和最佳標(biāo)記方法； 2.生物蛋白膠鋪于培養(yǎng)板底后植入標(biāo)記有BrdU的BMSCs體外成骨培養(yǎng)：成骨誘導(dǎo)后三組細(xì)胞形態(tài)由梭形變?yōu)榱⒎叫?細(xì)胞體積均增大,在成骨誘導(dǎo)后一周,開(kāi)始出現(xiàn)少量的細(xì)小的鈣結(jié)節(jié),隨著誘導(dǎo)時(shí)間的增長(zhǎng),鈣結(jié)節(jié)逐漸增大增多。同時(shí)間段C組(實(shí)驗(yàn)組)與A(對(duì)照組)、B (BrdU組)兩組相比,細(xì)胞貼壁率明顯.增高(P0.05)。行GENMED細(xì)胞堿性磷酸酶(ALP)活性染色和GENMED細(xì)胞馮庫(kù)薩(Von-Kossa)改良法染色,三組細(xì)胞隨培養(yǎng)時(shí)間增加,染色陽(yáng)性細(xì)胞和鈣結(jié)節(jié)逐漸增多。堿性磷酸酶及鈣定量檢測(cè),在同一時(shí)間段,C組(實(shí)驗(yàn)組)與A(對(duì)照組)、B (BrdU組)兩組相比均未見(jiàn)顯著差異,P0.05,無(wú)統(tǒng)計(jì)學(xué)意義。 3.標(biāo)記有BrdU的BMSCs直接融入生物蛋白膠中共同培養(yǎng)：骨髓基質(zhì)干細(xì)胞經(jīng)BrdU標(biāo)記后包埋于生物蛋白膠中能很好存活并增殖,3天后部分細(xì)胞呈短梭形狀,6天后生物蛋白膠邊緣部分開(kāi)始降解,細(xì)胞脫落致培養(yǎng)板；體外培養(yǎng)14天,細(xì)胞生長(zhǎng)良好,大部分生物蛋白膠降解,脫落的細(xì)胞增多,貼壁生長(zhǎng)的細(xì)胞形態(tài)正常；三周后生物蛋白膠完全降解。經(jīng)FDA-PI雙色熒光法檢測(cè)結(jié)果顯示,BMSCs活細(xì)胞率達(dá)95%以上,BMSCs在生物蛋白膠之中生長(zhǎng)良好。經(jīng)兩組細(xì)胞培養(yǎng)液中堿性磷酸酶(ALP)活性檢測(cè),實(shí)驗(yàn)組與對(duì)照組相比,未見(jiàn)明顯差異(P0.05),說(shuō)明生物蛋白膠包裹BMSCs后,對(duì)BMSCs成骨分化未見(jiàn)影響。 結(jié)論 1.BrdU標(biāo)記簡(jiǎn)單,快速,安全,檢測(cè)敏感性好,是反映細(xì)胞增殖及示蹤監(jiān)測(cè)移植細(xì)胞的理想指標(biāo)。 2.10umol/L標(biāo)記濃度及標(biāo)記48h是最佳標(biāo)記濃度和最佳標(biāo)記時(shí)間。 3.BMSCs植于生物蛋白膠表面培養(yǎng)后,細(xì)胞貼壁良好,說(shuō)明生物蛋白膠具有良好的組織相容性,對(duì)BMSCs未見(jiàn)明顯的的排斥現(xiàn)象。 4.生物蛋白膠在BMSCs作用下,具有良好的降解性,是組織工程中理想的細(xì)胞載體和移植的支架材料。 5.經(jīng)BrdU標(biāo)記的BMSCs包埋于生物蛋白膠所構(gòu)建的工程化組織后,其生長(zhǎng)、增殖及成骨分化等生物學(xué)特性沒(méi)有明顯不良影響,為進(jìn)一步體內(nèi)實(shí)驗(yàn)提供理論依據(jù)。
[Abstract]:Objective
The effect of 5- bromodeoxyuridine (BrdU) on bone marrow stromal cells (BMSCs) was studied to determine the best marker concentration and best marking time in vitro, and the effects of BrdU and FS on BMSCs growth, proliferation and osteogenic differentiation were observed by biological protein glue (FS) and BrdU labeled BMSCs in vitro. The effects of BrdU and FS on the growth of BMSCs, proliferation and osteogenic differentiation were observed. The feasibility of the scaffold as cell carrier provides experimental evidence for the application of Brdu labeled BMSCs/FS engineered tissue in vivo.
Method
1. in vitro isolation and culture of BMSCs and detection of the best concentration of BrdU markers and the best time: take 2 month old healthy New Zealand white rabbits, take out bilateral femur under aseptic state, use full bone marrow culture, use flow cytometry, identify the cell phenotype of CD34, CD44, CD90 and so on, and confirm that P3 generation BMSCs, with 1 x 105/ml cells after the high purity BMSCs. Density was inoculated in 16 pieces of 6 Hole culture plate (each culture plate was pre placed with glass slides in front of cell crawling slices), every 4 culture plates was one group, and four groups (A, B, C, D): L-DMEM full medium containing BrdU terminal concentration was 5umol/L, 10umol/L, 15umol/L, 20umol/L. The cell immuno histochemical staining of cell crawling slices in a culture plate was used to calculate the cell labeling rate and to compare the labeling rate of BrdU positive cells at different concentrations and at different time. The optimum concentration and optimal time of BrdU markers were determined.
2. biological protein gel was laid on the bottom of the culture plate and implanted into bone culture in vitro labeled with BrdU. The best BrdU marker concentration was used to mark the bone marrow stromal cells (Bone Mesenchymal stem cells BMSCs) with the above study. The experiment was divided into three groups: A group (control group): the unlabeled P3 generation BMSCs was added to the osteogenic induction culture medium; B group: (group BrdU): P3 BMSCs labeled with BrdU added to bone induced culture medium; C group: (experimental group): (experimental group): (experimental group): a thin layer of biological protein gel was laid on the bottom of culture plate, then BrdU BMSCs was inoculated and added to the osteogenic induction medium to avoid light culture. Three groups of cells were implanted in 24,96 hole culture plate according to the following detection purpose. 6 holes in the culture plate were selected at random as the number of detection. After the culture of 2,4,6,8,10,12h, the cell adherence rate was detected and the osteogenesis was induced for 1,2,4,7,14,21 days. The overall observation, growth kinetics (MTT), trypan blue staining, GENMED cell alkaline phosphatase (ALP) activity staining and GENMED cell von CO (Von-Kossa) were changed. Good staining method was also used for quantitative determination of alkaline phosphatase and quantitative determination of free calcium (methyl thymol blue colorimetric method).
3. BMSCs, labeled with BrdU, was directly integrated into the biological protein glue: P3 generation BMSCs marked with BrdU was selected and the cell count was counted after digestion, and about 1 x 107 BMSCs were mixed into the fibrinogen solution tube (about 5m1), and the BMSCs/FS complex marked with BrdU was formed after mixing with the liquid in the same amount of thrombin, which was added to the bottom of the culture plate. The L-DMEM complete culture medium was cultured in the experimental group, and the control group was added to the L-DMEM complete culture medium for the unmarked BrdU spoon P3 generation BMSCs implantation culture plate. The growth and proliferation of BMSCs in the biological protein gel in the experimental group were observed under the 1,3,7,14,21 sky light microscope, and the FDA PI double color fluorescence method was used to detect the living cell rate and judge BM. The survival rate of SCs in the biological protein glue; finally, the index of two groups of BMSCs to osteoblast differentiation: alkaline phosphatase (ALP) activity. In order to study the growth, proliferation and differentiation of BMSCs in the presence of BrdU and biological protein glue, whether the protein gum in vitro growth and proliferation of the bone marrow stromal cells labeled BrdU spoon. The presence of biodegradable protein and colloidal degradation.
The above data were analyzed by SPSS17.0 statistical software, and P0.05 was statistically significant.
1. in vitro isolation and culture of BMSCs and detection of the optimum concentration and optimum time for BrdU markers: P3 generation BMSCs through flow cytometry showed that the purity of bone marrow stromal stem cells was more than 90%.BrdU for nuclear labeling, and the nucleus marked brown red. At the marked concentration square, four groups of different concentrations in the same time point The labeling rate of 10umol/L, 15umol/L and 20umol/L was significantly higher than that of 5umol/L (P0.05), and there was no significant difference between 10umol/L, 15umol/L, 20umol/L concentration labeling ratio (P0.05). In the marking time, the four groups were significantly different in the same group 48h, 72h, and the 96h labeling rate. Compared with P0.05, there was no significant difference in the labeling rate of 48h, 72h and 96h in the same group. Therefore, it was indicated that the best marker concentration and best labeling method were 10umol/L concentration and marked 48h.
2. the 2. biological protein gel was laid on the bottom of the culture plate and implanted into the bone culture of BMSCs in vitro. After the induction of bone formation, the cell morphology of the three groups was cubic, the volume of the cells increased, and a small amount of fine calcium nodules began to appear in the week after the induction of osteogenesis. With the increase of induction time, the calcium nodules increased gradually. C group (experimental group) with A (control group) and B (group BrdU) two groups, the cell adherence rate was obvious. Increase (P0.05). GENMED cell alkaline phosphatase (ALP) activity staining and GENMED cell von CAA (Von-Kossa) improved staining, the three groups of cells with the increase of culture time, staining positive cells and calcium nodules gradually increased. Alkaline phosphatase and calcium quantitative detection At the same time, there was no significant difference between group C (experimental group) and A (control group) and B (BrdU group) compared with the two groups, P0.05 had no statistical significance.
3. BMSCs, which was labeled with BrdU, was directly integrated into the biological protein glue. The bone marrow stromal stem cells could survive and proliferate well in the biological protein glue after BrdU labeling. After 3 days, the cells showed a short shuttle shape. After 6 days, the marginal part of the biological protein gum began to degrade and the cells degenerated to the culture plate. The cells were cultured for 14 days in vitro and the cells grew well. Well, most of the biological protein gelatin was degraded, the cells were increased, the cell morphology of the adherent growth was normal, and the biological protein glue was completely degraded after three weeks. The results of FDA-PI double color fluorescence detection showed that the rate of BMSCs living cells was above 95%, and BMSCs grew well in the biological protein glue. The alkaline phosphatase (ALP) activity in the two groups of cell culture fluid There was no significant difference between the experimental group and the control group (P0.05), indicating that the biological protein adhesive encapsulated BMSCs had no effect on the osteogenic differentiation of BMSCs.
conclusion
The 1.BrdU marker is simple, rapid, safe and sensitive to detection. It is an ideal index for monitoring cell proliferation and tracing the monitoring of transplanted cells.
2.10umol/L marker concentration and labeled 48h were the best marker concentration and the best labeling time.
After the 3.BMSCs was cultivated on the surface of the protein glue, the cell adhered well, indicating that the biological protein gum had good histocompatibility and no obvious rejection to the BMSCs.
4. biological protein adhesive has good degradability under the action of BMSCs. It is an ideal cell carrier and scaffold material for tissue engineering.
5. BrdU labeled BMSCs was embedded in the engineered tissue constructed by biological protein gel, and the biological characteristics of its growth, proliferation and osteogenic differentiation had no obvious adverse effects, which provided a theoretical basis for further study in vivo.
【學(xué)位授予單位】：昆明醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R329

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