【摘要】： 目的:對(duì)體外培養(yǎng)C57小鼠密質(zhì)骨來源的間充質(zhì)祖細(xì)胞(mesenchymal progenitor cells,MPC)的方法進(jìn)行研究,建立適合小鼠MPC增殖的培養(yǎng)方案;探討小鼠密質(zhì)骨來源的MPC體外向神經(jīng)元樣細(xì)胞定向誘導(dǎo)分化的實(shí)驗(yàn)研究。 方法: 取材方法選擇健康C57雌性小鼠,清潔級(jí),3周齡,18±2 g,取股骨、脛骨碎片經(jīng)Ⅱ型膠原蛋白酶消化后作為MPC來源。 分組取得消化后的骨碎片,選取干細(xì)胞培養(yǎng)中經(jīng)常使用的DMEM/F12培養(yǎng)基、IMDM培養(yǎng)基、α-MEM培養(yǎng)基和TBD公司生產(chǎn)的胎牛血清(FCS)、GIBCO公司生產(chǎn)的胎牛血清,按不同搭配進(jìn)行培養(yǎng),隨機(jī)分為DMEM/F12+10%TBD胎牛血清組、DMEM/F12 +10%GIBCO胎牛血清組、IMDM+10%TBD胎牛血清組、IMDM +10%GIBCO胎牛血清組、α-MEM +10%TBD胎牛血清組、α-MEM +10%GIBCO胎牛血清組,共6組,每組培養(yǎng)6份。 培養(yǎng)方法將小鼠密質(zhì)骨碎片置入六孔板中,按不同分組加入相應(yīng)培養(yǎng)液,放入37℃、5% CO2的培養(yǎng)箱內(nèi),48 h后首次換液,以后每隔2-3 d換液一次。每天觀察細(xì)胞生長情況,當(dāng)生長最好的實(shí)驗(yàn)組原代(P0)細(xì)胞細(xì)胞貼壁超過培養(yǎng)板底面積80%時(shí)吸出培養(yǎng)液,用含0.04% EDTA的0.25%胰蛋白酶消化、收集細(xì)胞,按固定細(xì)胞濃度傳入培養(yǎng)瓶。傳代后以后每2-3 d換液一次,當(dāng)生長最好的實(shí)驗(yàn)組細(xì)胞貼壁超過培養(yǎng)瓶底面積70%-80%時(shí)按固定細(xì)胞濃度1×103個(gè)/cm2傳代。 MPC純度鑒定采用流式細(xì)胞術(shù)分析,單標(biāo)法檢測第三代(P3)MPC,取FITC標(biāo)記的兔抗鼠CD29、CD31、CD44、CD90和PE標(biāo)記的兔抗鼠CD45、CD106作為標(biāo)記物,平行對(duì)照組為加入同型對(duì)照抗體的MPC�？紤]到實(shí)驗(yàn)的需要以及研究生學(xué)習(xí)階段時(shí)間有限,只選擇采用優(yōu)化方案進(jìn)行培養(yǎng)的MPC進(jìn)行純度鑒定。 MPC的多向分化鑒定對(duì)MPC進(jìn)行向骨細(xì)胞、脂肪細(xì)胞的定向誘導(dǎo)分化,驗(yàn)證其具有干細(xì)胞的多向分化潛能,用茜素紅和油紅O染色檢測誘導(dǎo)分化結(jié)果。同樣只選擇采用優(yōu)化方案進(jìn)行培養(yǎng)的MPC進(jìn)行多向分化能力鑒定。 MPC定向誘導(dǎo)成神經(jīng)元樣細(xì)胞的方法取優(yōu)化方案培養(yǎng)的P4代MPC,用微環(huán)境體液(神經(jīng)元原代培養(yǎng)上清液)進(jìn)行誘導(dǎo),取誘導(dǎo)24 h后的MPC作為實(shí)驗(yàn)標(biāo)本。對(duì)其進(jìn)行形態(tài)學(xué)觀察;免疫細(xì)胞化學(xué)檢測神經(jīng)元特異性標(biāo)志物神經(jīng)元特異性烯醇化酶(neuron specific enolase ,NSE)及神經(jīng)絲蛋白(neurofilament protein,NF)的表達(dá)情況,同時(shí)進(jìn)行免疫熒光分析。 結(jié)果: C57小鼠間充質(zhì)祖細(xì)胞(MPC)體外優(yōu)化培養(yǎng) 原代培養(yǎng)5 d后,可見培養(yǎng)器皿底部有細(xì)胞貼壁生長,以圓形和扁圓形為主,10 d后貼壁細(xì)胞逐漸鋪開,形成梭形或多角形,形態(tài)飽滿,折光性強(qiáng),細(xì)胞間無重疊,有接觸抑制現(xiàn)象;傳代后細(xì)胞形態(tài)趨于一致,大部分為梭形,排列緊密,生長旺盛。當(dāng)某個(gè)實(shí)驗(yàn)組細(xì)胞貼壁超過底面積70%-80%時(shí),對(duì)所有細(xì)胞進(jìn)行傳代計(jì)數(shù),觀察3代。結(jié)果發(fā)現(xiàn)在每一代的細(xì)胞計(jì)數(shù)中,DMEM/F12+10% GIBCO胎牛血清組的細(xì)胞數(shù)量都超過其他各組,通過統(tǒng)計(jì)學(xué)檢驗(yàn)驗(yàn)證DMEM/F12+10% GIBCO胎牛血清組細(xì)胞數(shù)量與其余各組相比有顯著差異(P㩳0.05)。說明DMEM/F12+10%GIBCO胎牛血清更有利于MPC的體外培養(yǎng)增殖。 MPC純度鑒定 優(yōu)化方案培養(yǎng)的MPC表達(dá)間質(zhì)細(xì)胞標(biāo)記CD29、CD44、CD90和CD106,不表達(dá)造血細(xì)胞標(biāo)記CD31和CD45。說明得到的是同源性好、純度高、排除了造血干細(xì)胞影響的MPC。 MPC多向分化能力鑒定 在用優(yōu)化方案培養(yǎng)的MPC定向誘導(dǎo)成骨細(xì)胞的實(shí)驗(yàn)中,通過茜素紅染色發(fā)現(xiàn)誘導(dǎo)后細(xì)胞外基質(zhì)中大量鈣鹽沉積;在定向誘導(dǎo)成脂肪細(xì)胞的實(shí)驗(yàn)中,通過油紅O染色發(fā)現(xiàn)細(xì)胞中出現(xiàn)的脂滴被染成點(diǎn)狀紅色,表明我們得到的MPC能向骨細(xì)胞、脂肪細(xì)胞分化。說明我們得到的是活性好、具有多向分化能力的MPC。 MPC體外向神經(jīng)元樣細(xì)胞定向誘導(dǎo)分化 經(jīng)誘導(dǎo)24 h后,MPC細(xì)胞形態(tài)發(fā)生變化,自胞體有突起長出,各細(xì)胞突起長短不一,類似神經(jīng)元。免疫細(xì)胞化學(xué)檢測結(jié)果表明,誘導(dǎo)后MPC的NSE(73.73%±9.88%)及NF(60.26%±7.19%)均陽性表達(dá);免疫熒光檢測也證實(shí)免疫細(xì)胞化學(xué)檢測的結(jié)果。 結(jié)論 1.實(shí)驗(yàn)證明,用DMEM/F12+10% GIBCO胎牛血清為培養(yǎng)液進(jìn)行小鼠密質(zhì)骨來源的MPC體外培養(yǎng)最有利于其數(shù)量的擴(kuò)增,得到的是同源性好、純度高、增殖活力強(qiáng)、具有多向分化潛能的MPC細(xì)胞群。 2.通過微環(huán)境體液(神經(jīng)元原代培養(yǎng)上清液)誘導(dǎo)的方法能夠使小鼠密質(zhì)骨來源的MPC定向分化為神經(jīng)元樣細(xì)胞。
[Abstract]:AIM: To study the method of culturing mesenchymal progenitor cells (MPCs) derived from mouse compact bone in vitro and establish a culture scheme suitable for the proliferation of mouse MPCs.
Method:
Methods Healthy C57 female mice, clean grade, 3 weeks old, 18 + 2 g, were selected as MPC source.
Digested bone fragments were obtained by grouping. DMEM/F12 medium, IMDM medium, alpha-MEM medium, fetal bovine serum (FCS) produced by TBD company and fetal bovine serum produced by GIBCO company were selected and cultured in different combinations. They were randomly divided into DMEM/F12+10% TBD fetal bovine serum group, DMEM/F12+10% GIBCO fetal bovine serum group, IM/F12+10% GIBCO fetal bovine serum group. DM+10% TBD fetal bovine serum group, IMDM+10% GIBCO fetal bovine serum group, alpha-MEM+10% TBD fetal bovine serum group, alpha-MEM+10% GIBCO fetal bovine serum group, a total of 6 groups, each group culture 6.
Methods The dense bone fragments of mice were put into six-hole plate, and the corresponding culture medium was added according to different groups. The culture medium was first changed after 48 hours, and then changed every 2-3 days. The culture medium was digested with 0.25% trypsin containing 0.04% EDTA. The cells were collected and passed into the culture flask at fixed cell concentration. After passage, the culture medium was changed every 2-3 days. When the adherence area of the best growing experimental group exceeded 70% -80% of the bottom area of the culture flask, the cells were subcultured at fixed cell concentration of 1 *103 cells per cm 2.
The purity of MPC was determined by flow cytometry. The third generation (P3) MPC was detected by single-label method. The FITC-labeled rabbit anti-mouse CD29, CD31, CD44, CD90 and PE-labeled rabbit anti-mouse CD45 and CD106 were used as markers. The parallel control group was used as MPC with homologous control antibodies. The purity of MPC was determined by the culture.
MPC was differentiated into osteoblasts and adipocytes by directional induction. The differentiation potential of MPC was verified by alizarin red and oil red O staining.
MPC was induced into neuron-like cells by microenvironment humor (supernatant of primary culture of neurons) and 24 hours after induction. Morphological observation and immunocytochemical detection of neuron-specific marker neuron-specific enolase (neuron-specific enolase) were performed. The expression of specific enolase, NSE and neurofilament protein (NF) was analyzed by immunofluorescence.
Result:
In vitro optimization culture of C57 mouse mesenchymal progenitor cells (MPC)
After primary culture for 5 days, the cells adhered to the wall and grew mainly round and oblate. After 10 days, the adherent cells gradually spread out, forming spindle or polygonal shape, full shape, strong refraction, no overlap between cells, and contact inhibition phenomenon. After passage, the cell morphology tended to be consistent, most of them were spindle-shaped, arranged closely, and grew vigorously. The number of cells in DMEM/F12+10% GIBCO fetal bovine serum group was higher than that in other groups in each generation. The number of cells in DMEM/F12+10% GIBCO fetal bovine serum group and other groups were verified by statistical test. The results showed that DMEM/F12+10% GIBCO fetal bovine serum was more conducive to the proliferation of MPC in vitro.
Purity identification of MPC
MPC cultured in the optimized scheme expressed interstitial cell markers CD29, CD44, CD90 and CD106, but did not express hematopoietic cell markers CD31 and CD45.
MPC identification of multiple differentiation ability
In the experiment of directional induction of osteoblasts by MPC cultured with optimized scheme, a large amount of calcium salt was found in the extracellular matrix after induction by alizarin red staining; in the experiment of directional induction of adipocytes, fat droplets were found to be dotted red by oil red O staining, indicating that the obtained MPC could be directed to osteoblasts, lipids. The differentiation of cells shows that we have obtained MPC. with good activity and multiple differentiation ability.
In vitro differentiation of MPC into neuron like cells in vitro
After 24 hours of induction, the morphology of MPC cells changed, and the processes grew out of the cell body. The length of each cell process was different, which was similar to neurons.
conclusion
1. Experiments showed that DMEM/F12+10% GIBCO fetal bovine serum was the best medium for the expansion of MPC derived from mouse compact bone in vitro. The MPC cells with good homology, high purity, strong proliferative activity and multi-differentiation potential were obtained.
2. Microenvironment bodily fluid (supernatant of primary culture of neurons) can induce mouse compact bone-derived MPCs to differentiate into neuron-like cells.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R329

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