發(fā)布時(shí)間：2018-06-30 07:50

  本文選題：骨髓間充質(zhì)干細(xì)胞 + 成骨生長太(OGP)��； 參考：《昆明醫(yī)科大學(xué)》2013年碩士論文

【摘要】：目的： 探討研究成骨生長肽(OGP)對兔骨髓間充質(zhì)干細(xì)胞體外培養(yǎng)增殖及成骨分化的影響并探討骨髓間充質(zhì)干細(xì)胞在OGP濃度10-11-10-7mol/L范圍內(nèi)的細(xì)胞最適宜增殖濃度以及最適宜成骨分化濃度,為成骨生長肽(OGP)進(jìn)一步實(shí)驗(yàn)室研究及體內(nèi)研究提供可靠的理論依據(jù) 方法： 取達(dá)到清潔級標(biāo)準(zhǔn),無人畜共患疾病的新西蘭大白兔一只,不限雌雄,使用兔耳緣靜脈空氣栓塞發(fā)處死。嚴(yán)格遵守?zé)o菌原則取出雙側(cè)股骨干。使用DMEM/F12培養(yǎng)液沖洗髓腔,獲得細(xì)胞懸濁液。應(yīng)用全骨髓培養(yǎng)法分離出骨髓單核細(xì)胞,使用貼壁法可以除去沒有貼壁的懸浮細(xì)胞。經(jīng)過反復(fù)多次的傳代培養(yǎng)能夠獲得較為純凈的貼壁細(xì)胞,使用免疫組化檢測貼壁細(xì)胞的表面標(biāo)記物CD44,可以證實(shí)所得的細(xì)胞為骨髓間充質(zhì)干細(xì)胞。 貼壁法分離獲得的兔骨髓間充質(zhì)干細(xì)胞,通過換液和傳代純化細(xì)胞。用傳二代細(xì)胞做MTT試驗(yàn),以此確定OGP對骨髓間充質(zhì)干細(xì)胞增殖作用的最佳活性濃度；將傳二代骨髓間充質(zhì)干細(xì)胞,以2×104/m1的密度接種于96孔培養(yǎng)板中,每孔加入200u1細(xì)胞懸濁液。一共接種7板,每組接種6孔,每板接種36孔。分為對照組與實(shí)驗(yàn)組,具體如下：分為六組：A組-對照組,B組加入10-11mol/L的OGP,C組加入10-10mol/L的OGP,D組加入10-9mol/L的OGP,E組加入10-8mol/L的OGP,F組10-7mol/L的OGP。對照組為不含血清的DMEM/F12培養(yǎng)基。 將傳3代骨髓間充質(zhì)干細(xì)胞接種于16孔板中,每組接種4孔,共獲取16個(gè)樣品。分別于第五天,十天,十五天,二十天四個(gè)時(shí)間點(diǎn)收集。每一時(shí)間點(diǎn)設(shè)置6個(gè)復(fù)孔分別檢測堿性磷酸酶的活性,以確定促進(jìn)骨髓間充質(zhì)干細(xì)胞成骨分化最佳OGP活性濃度。分組具體如下：A組培養(yǎng)液為50μg/ml維生素C+10mmol/L β-甘油磷酸鈉+10-7mol/LOGP；B組培養(yǎng)液為50μg/ml維生C+10mmol/L β-甘油磷酸鈉+10-9mol/LOGP；C組培養(yǎng)液50μ g/ml維生素C+10mmol/L β-甘油磷酸鈉+10-11mol/LOGP;D組(對照組)培養(yǎng)50μg/ml維生素C+10mmol/Lβ甘油磷酸鈉。 在最適宜骨髓間充質(zhì)干細(xì)胞成骨分化的OGP濃度培養(yǎng)下的骨髓間充質(zhì)干細(xì)胞的細(xì)胞形態(tài)學(xué)觀察,以及對其進(jìn)行堿性磷酸酶(ALP)Gomori鈣鈷染色,Von kossa改良染色,和MTB比色法進(jìn)行鈣定量檢測。分為兩組：A組對照組單純培養(yǎng)液組；B組加入OGP的培養(yǎng)液組。 結(jié)果 骨髓間充質(zhì)干細(xì)胞分離培養(yǎng)后最初培養(yǎng)器中造血細(xì)胞成份較多,隨著時(shí)間的延長,這些雜質(zhì)細(xì)胞慢慢壞死或隨換液移去,2日后能夠見到部分細(xì)胞開始貼壁生長,大約4日后可見大量細(xì)胞貼壁,成集落,細(xì)胞開始呈長梭形,形似成纖維細(xì)胞,呈放射狀生長。培養(yǎng)至12天左右,細(xì)胞融合超過90%予以傳代,培養(yǎng)到第3代后,骨髓間充質(zhì)干細(xì)胞就較純粹,極少雜質(zhì)細(xì)胞。對培養(yǎng)細(xì)胞用免疫組化進(jìn)行分析,發(fā)現(xiàn)骨髓間充質(zhì)干細(xì)胞的特異性抗體CD44(+)。證明培養(yǎng)的細(xì)胞為骨髓間充質(zhì)干細(xì)胞。 將成骨誘導(dǎo)培養(yǎng)液加入至骨髓間充質(zhì)干細(xì)胞的培養(yǎng)后,細(xì)胞生長表現(xiàn)為會有一個(gè)較長的生長平臺期約一天至兩天,此時(shí)細(xì)胞呈緩慢生長,于此同時(shí)細(xì)胞形態(tài)也大多發(fā)生改變,我們觀察到細(xì)胞慢慢由紡錘形漸漸轉(zhuǎn)變?yōu)榱⒎叫?然后變更為多邊形,細(xì)胞體積變大,胞漿邊濃密；對細(xì)胞進(jìn)行消化傳代培養(yǎng),傳代到P3代時(shí),細(xì)胞會經(jīng)過一長約24到48小時(shí)左右的潛伏期,在將近第150到200小時(shí)左右時(shí),細(xì)胞度過平臺期,開始呈對數(shù)生長,后仍會過渡到平臺期。 MTT染色法測定OGP的最大活性濃度：骨髓間充質(zhì)干細(xì)胞在含有OGP的培養(yǎng)基中生長,光吸收度數(shù)值均比其對照組(無OGP的培養(yǎng)基)中的光吸收度數(shù)值要高。當(dāng)培養(yǎng)基中的OGP濃度為10-11mol/L時(shí),可以獲得最積極的細(xì)胞生長增殖。 堿性磷酸酶測定骨髓間充質(zhì)干細(xì)胞活化的最適宜OGP的濃度：D組(對照組)中堿性磷酸酶的表達(dá)一直處于較低水平的表達(dá)。與其他組相比,其余組的ALP表達(dá)顯著升高。而A,B,C組中又以B組(OGP濃度為10-9mol/L)的堿性磷酸酶活性表達(dá)最高。 對于內(nèi)含10-9mol/L的GOP的培養(yǎng)液對骨髓間充值干細(xì)胞成骨誘導(dǎo)的形態(tài)學(xué)觀察,堿性磷酸酶(ALP) Gomori鈣鈷染色法,Von Kossa改良然染色法,以及MTB比色法進(jìn)行鈣定量檢測： 第一應(yīng)用10-9mol/L的OGP的培養(yǎng)液后細(xì)胞漸漸由紡錘形轉(zhuǎn)變?yōu)榱⒎叫?在變?yōu)槎噙呅?細(xì)胞表面積變大,細(xì)胞核、質(zhì)分界清楚,細(xì)胞漿濃密,細(xì)胞呈階層是增長,未見接觸抑制,形成廣泛分布的細(xì)胞結(jié)節(jié)。 第二堿性磷酸酶(ALP)Gomori鈣鈷染色法,于誘導(dǎo)后第14天至21天時(shí)發(fā)現(xiàn)：堿性磷酸酶在由大多數(shù)弱陽性或陰性,少數(shù)強(qiáng)陽性逐漸轉(zhuǎn)變成幾乎全部的細(xì)胞均呈現(xiàn)出強(qiáng)陽性。并且細(xì)胞質(zhì)中隨處可見黑色顆�；驂K狀沉淀。而對照組無陽性表現(xiàn)出現(xiàn)。 第三Von Kossa改良然染色法測定鈣的沉積,同樣于誘導(dǎo)后第14天至21天時(shí)發(fā)現(xiàn)：逐漸增多,顏色加重的結(jié)節(jié)。由棕黑色逐漸變?yōu)楹谏?并且越來越多,越變越大。而對照組無此變化。 第四MTB比色法進(jìn)行鈣定量檢測,在第5天,10天,15天,20天分別使用MTB比色法測定,在含有OGP (10-9mol/L)培養(yǎng)基培養(yǎng)下的骨髓間充質(zhì)干細(xì)胞成骨誘導(dǎo)明顯高于對照組(無OGP)。方差統(tǒng)計(jì)分析算得p0.05,具有統(tǒng)計(jì)學(xué)意義。 結(jié)論 1.進(jìn)一步證實(shí)了OGP對兔骨髓間充質(zhì)干細(xì)胞的增殖活性有明顯的促進(jìn)作用,并且最佳作用濃度為10-11mol/L,在(10-7-10-11mol/L)濃度范圍內(nèi)存在負(fù)相關(guān)量效關(guān)系 2.OGP促進(jìn)骨髓間充質(zhì)干細(xì)胞的ALP表達(dá)的最佳濃度為10-9mol/L.并且所有添加了OGP的培養(yǎng)基培養(yǎng)的骨髓間充質(zhì)干細(xì)胞的成骨誘導(dǎo)均優(yōu)于普通礦物培養(yǎng)基培養(yǎng)的骨髓間充質(zhì)干細(xì)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.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R329

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