OGP对兔骨髓间充质干细胞体外成骨分化干预的实验研究

发布时间：2018-06-30 07:50

本文选题：骨髓间充质干细胞 + 成骨生长太(OGP)　；参考：《昆明医科大学》2013年硕士论文

【摘要】：目的：探讨研究成骨生长肽(OGP)对兔骨髓间充质干细胞体外培养增殖及成骨分化的影响并探讨骨髓间充质干细胞在OGP浓度10-11-10-7mol/L范围内的细胞最适宜增殖浓度以及最适宜成骨分化浓度,为成骨生长肽(OGP)进一步实验室研究及体内研究提供可靠的理论依据方法：取达到清洁级标准,无人畜共患疾病的新西兰大白兔一只,不限雌雄,使用兔耳缘静脉空气栓塞发处死。严格遵守无菌原则取出双侧股骨干。使用DMEM/F12培养液冲洗髓腔,获得细胞悬浊液。应用全骨髓培养法分离出骨髓单核细胞,使用贴壁法可以除去没有贴壁的悬浮细胞。经过反复多次的传代培养能够获得较为纯净的贴壁细胞,使用免疫组化检测贴壁细胞的表面标记物CD44,可以证实所得的细胞为骨髓间充质干细胞。贴壁法分离获得的兔骨髓间充质干细胞,通过换液和传代纯化细胞。用传二代细胞做MTT试验,以此确定OGP对骨髓间充质干细胞增殖作用的最佳活性浓度；将传二代骨髓间充质干细胞,以2×104/m1的密度接种于96孔培养板中,每孔加入200u1细胞悬浊液。一共接种7板,每组接种6孔,每板接种36孔。分为对照组与实验组,具体如下：分为六组：A组-对照组,B组加入10-11mol/L的OGP,C组加入10-10mol/L的OGP,D组加入10-9mol/L的OGP,E组加入10-8mol/L的OGP,F组10-7mol/L的OGP。对照组为不含血清的DMEM/F12培养基。将传3代骨髓间充质干细胞接种于16孔板中,每组接种4孔,共获取16个样品。分别于第五天,十天,十五天,二十天四个时间点收集。每一时间点设置6个复孔分别检测碱性磷酸酶的活性,以确定促进骨髓间充质干细胞成骨分化最佳OGP活性浓度。分组具体如下：A组培养液为50μg/ml维生素C+10mmol/L β-甘油磷酸钠+10-7mol/LOGP；B组培养液为50μg/ml维生C+10mmol/L β-甘油磷酸钠+10-9mol/LOGP；C组培养液50μ g/ml维生素C+10mmol/L β-甘油磷酸钠+10-11mol/LOGP;D组(对照组)培养50μg/ml维生素C+10mmol/Lβ甘油磷酸钠。在最适宜骨髓间充质干细胞成骨分化的OGP浓度培养下的骨髓间充质干细胞的细胞形态学观察,以及对其进行碱性磷酸酶(ALP)Gomori钙钴染色,Von kossa改良染色,和MTB比色法进行钙定量检测。分为两组：A组对照组单纯培养液组；B组加入OGP的培养液组。结果骨髓间充质干细胞分离培养后最初培养器中造血细胞成份较多,随着时间的延长,这些杂质细胞慢慢坏死或随换液移去,2日后能够见到部分细胞开始贴壁生长,大约4日后可见大量细胞贴壁,成集落,细胞开始呈长梭形,形似成纤维细胞,呈放射状生长。培养至12天左右,细胞融合超过90%予以传代,培养到第3代后,骨髓间充质干细胞就较纯粹,极少杂质细胞。对培养细胞用免疫组化进行分析,发现骨髓间充质干细胞的特异性抗体CD44(+)。证明培养的细胞为骨髓间充质干细胞。将成骨诱导培养液加入至骨髓间充质干细胞的培养后,细胞生长表现为会有一个较长的生长平台期约一天至两天,此时细胞呈缓慢生长,于此同时细胞形态也大多发生改变,我们观察到细胞慢慢由纺锤形渐渐转变为立方形,然后变更为多边形,细胞体积变大,胞浆边浓密；对细胞进行消化传代培养,传代到P3代时,细胞会经过一长约24到48小时左右的潜伏期,在将近第150到200小时左右时,细胞度过平台期,开始呈对数生长,后仍会过渡到平台期。 MTT染色法测定OGP的最大活性浓度：骨髓间充质干细胞在含有OGP的培养基中生长,光吸收度数值均比其对照组(无OGP的培养基)中的光吸收度数值要高。当培养基中的OGP浓度为10-11mol/L时,可以获得最积极的细胞生长增殖。碱性磷酸酶测定骨髓间充质干细胞活化的最适宜OGP的浓度：D组(对照组)中碱性磷酸酶的表达一直处于较低水平的表达。与其他组相比,其余组的ALP表达显著升高。而A,B,C组中又以B组(OGP浓度为10-9mol/L)的碱性磷酸酶活性表达最高。对于内含10-9mol/L的GOP的培养液对骨髓间充值干细胞成骨诱导的形态学观察,碱性磷酸酶(ALP) Gomori钙钴染色法,Von Kossa改良然染色法,以及MTB比色法进行钙定量检测：第一应用10-9mol/L的OGP的培养液后细胞渐渐由纺锤形转变为立方形,在变为多边形,细胞表面积变大,细胞核、质分界清楚,细胞浆浓密,细胞呈阶层是增长,未见接触抑制,形成广泛分布的细胞结节。第二碱性磷酸酶(ALP)Gomori钙钴染色法,于诱导后第14天至21天时发现：碱性磷酸酶在由大多数弱阳性或阴性,少数强阳性逐渐转变成几乎全部的细胞均呈现出强阳性。并且细胞质中随处可见黑色颗粒或块状沉淀。而对照组无阳性表现出现。第三Von Kossa改良然染色法测定钙的沉积,同样于诱导后第14天至21天时发现：逐渐增多,颜色加重的结节。由棕黑色逐渐变为黑色,并且越来越多,越变越大。而对照组无此变化。第四MTB比色法进行钙定量检测,在第5天,10天,15天,20天分别使用MTB比色法测定,在含有OGP (10-9mol/L)培养基培养下的骨髓间充质干细胞成骨诱导明显高于对照组(无OGP)。方差统计分析算得p0.05,具有统计学意义。结论 1.进一步证实了OGP对兔骨髓间充质干细胞的增殖活性有明显的促进作用,并且最佳作用浓度为10-11mol/L,在(10-7-10-11mol/L)浓度范围内存在负相关量效关系 2.OGP促进骨髓间充质干细胞的ALP表达的最佳浓度为10-9mol/L.并且所有添加了OGP的培养基培养的骨髓间充质干细胞的成骨诱导均优于普通矿物培养基培养的骨髓间充质干细胞。
[Abstract]:Objective:
The effect of osteogenic growth peptide (OGP) on the proliferation and osteogenic differentiation of rabbit bone marrow mesenchymal stem cells in vitro and the optimal concentration of bone marrow mesenchymal stem cells in the range of OGP concentration 10-11-10-7mol/L and the optimum concentration of osteogenic differentiation for osteogenic peptide (OGP) in the laboratory and in vivo Provide a reliable theoretical basis
Method:
A New Zealand white rabbit, unrestricted animal and male, was executed with an air embolism in the ear edge vein of a rabbit. The DMEM/F12 culture solution was used to rinse the marrow cavity and obtain the cell suspension. The bone marrow mononuclear cells were separated by the whole bone marrow culture method, and the adherent wall was used. The method can remove the non adherent cell suspension cells. After repeated passages, more purified adherent cells can be obtained, and the surface marker CD44 of the adherent cells can be detected by immunohistochemistry. It can be proved that the cells are bone marrow mesenchymal stem cells.
The rabbit bone marrow mesenchymal stem cells were separated by adhesion method, and the cells were purified by exchange and passage. The best activity concentration of OGP on the proliferation of bone marrow mesenchymal stem cells was determined with the transmission of two generation cells. The two generation bone marrow mesenchymal stem cells were inoculated in 96 hole culture plate with the density of 2 x 104/m1, and each hole was added to 200u1. A total of 7 plates were inoculated into 7 plates. Each group was inoculated 6 holes and each plate was inoculated with 36 holes. The control group and the experimental group were divided into the control group and the experimental group. The following were divided into six groups: the A group - the control group, the group B, the OGP, the OGP of the 10-10mol/L, the D group adding 10-9mol/L OGP, the E group joining the 10-8mol/L F12 medium.
The 3 generation bone marrow mesenchymal stem cells were inoculated into 16 orifice plates. Each group was inoculated with 4 holes, and 16 samples were obtained. They were collected at fifth days, ten days, fifteen days, twenty days and four time points respectively. The activity of alkaline phosphatase was detected by 6 complex holes at each time point to determine the best OGP activity to promote bone marrow mesenchymal stem cells differentiation. The results were as follows: the culture medium of group A was 50 mu g/ml vitamin C+10mmol/L beta glycerphosphate +10-7mol/LOGP, and the culture solution of B group was 50 UG C+10mmol/L beta glycerphosphate sodium +10-9mol/LOGP, and C group culture medium 50 micron C+10mmol/L beta glycerphosphate sodium +10-11mol/LOGP; 50 micron vitamins were cultivated in the group (control group). /L beta glycerol phosphate.
The morphological observation of bone marrow mesenchymal stem cells under OGP concentration suitable for bone differentiation of bone marrow mesenchymal stem cells, as well as alkaline phosphatase (ALP) Gomori calcium cobalt staining, Von Kossa dyeing, and MTB colorimetric method for quantitative determination of calcium, divided into two groups: A group control group simple culture group; B group adding OGP. The culture fluid group.
Result
After the separation and culture of bone marrow mesenchymal stem cells, there were more hematopoietic cells in the initial culture. With the prolongation of time, these cells were slowly necrotic or removed with the fluid. After 2 days, some cells began to adhere to the wall. After 4 days, a large number of cells were adhered to the cells, and the cells began to form a long shuttle, like fibroblasts. After 12 days, the cells were cultured for about 12 days, and the cells fused more than 90% to be subcultured. After the culture to third generations, the bone marrow mesenchymal stem cells were pure and very few. The specific antibody (+) of bone marrow mesenchymal stem cells was detected by immunohistochemistry. The cultured cells were bone marrow mesenchymal stem cells.
After the osteogenic induction culture is added to the culture of bone marrow mesenchymal stem cells, the cell growth shows that there will be a longer growth platform from about one to two days. At this time, the cells grow slowly. At the same time, the cell morphology is mostly changed. We observed that the cells gradually changed from spindle shape to cubic, and then changed into The cell volume becomes larger and the cytosolic edge is dense; the cells are digested and passed on to the P3 generation, and the cells pass through the incubation period of about 24 to 48 hours. When the cells are about 150th to 200 hours, the cells pass the platform period and begin to grow logarithmically, and then they will still transition to the platform stage.
The maximum activity concentration of OGP was determined by MTT staining: bone marrow mesenchymal stem cells were grown in the medium containing OGP, and the optical absorbance values were higher than those of the control group (no OGP medium). When the concentration of OGP in the medium was 10-11mol/L, the most active cell growth and proliferation could be obtained.
The most suitable OGP concentration of alkaline phosphatase in bone marrow mesenchymal stem cells activation: the expression of alkaline phosphatase in group D (control group) was always at a lower level. Compared with other groups, the expression of ALP in other groups increased significantly. The activity of alkaline phosphatase in the group of A, B and C was the highest in the B group (OGP concentration of 10-9mol/L).
The morphological observation of osteogenesis induced by bone marrow revalued stem cells in the medium containing 10-9mol/L containing GOP, alkaline phosphatase (ALP) Gomori calcium cobalt staining, Von Kossa modified colorimetry, and MTB colorimetric assay for quantitative determination of calcium.
After the first application of the culture fluid of OGP in 10-9mol/L, the cells gradually changed from spindle shape to cubic shape, the cell surface area became larger, the cell surface area became larger, the nucleus, the mass boundary was clear, the cell slurry was dense, the cell was growing, no contact inhibition was found, and the widely distributed cell nodules were formed.
Second alkaline phosphatase (ALP) Gomori calcium cobalt staining method was found after fourteenth days to 21 days after induction. Alkaline phosphatase was found to be strongly positive by the majority of weak positive or negative, and a few strong positive cells were gradually transformed into almost all cells, and black particles or massive precipitates were found everywhere in the cytoplasm. The control group did not show positive expression. Now.
Third Von Kossa modified blue staining method was used to determine the deposition of calcium. It was also found from fourteenth days to 21 days after induction: gradually increasing, color aggravated nodules gradually changed from brown black to black, and more and more, the greater the change, but no change in the control group.
Fourth MTB colorimetric assay was used for quantitative determination of calcium in fifth days, 10 days, 15 days and 20 days respectively. The osteogenic induction of bone marrow mesenchymal stem cells in the culture medium containing OGP (10-9mol/L) culture was significantly higher than that of the control group (no OGP). The statistical analysis of variance was P0.05, and the statistical significance was statistically significant.
conclusion
1. further confirmed that OGP has an obvious promoting effect on the proliferation activity of rabbit bone marrow mesenchymal stem cells, and the optimum concentration is 10-11mol/L, and in the concentration range of (10-7-10-11mol/L), the memory is negatively correlated.
The optimal concentration of 2.OGP to promote the expression of ALP in bone marrow mesenchymal stem cells is 10-9mol/L. and the osteogenic induction of all bone marrow mesenchymal stem cells cultured in the medium of OGP is superior to that of bone marrow mesenchymal stem cells cultured in ordinary mineral medium.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R329

【参考文献】