本文選題：次聲 + 骨髓間充質(zhì)干細(xì)胞(BMSCs)增殖��； 參考：《南方醫(yī)科大學(xué)》2012年碩士論文

【摘要】：研究背景 干細(xì)胞的研究和應(yīng)用是重大疾病再生性修復(fù)和治療的新途徑和新希望。目前關(guān)于干細(xì)胞的分離、鑒定、培養(yǎng)、擴(kuò)增、保存、復(fù)蘇等關(guān)鍵技術(shù)得到了各個(gè)研究機(jī)構(gòu)及個(gè)人的高度關(guān)注,而BMSCs因其來源充足、可實(shí)現(xiàn)自體移植等優(yōu)點(diǎn)倍受青睞。 次聲波是頻率在0.0001～20Hz范圍內(nèi)的機(jī)械振動波,可能有機(jī)械效應(yīng)、溫?zé)嵝?yīng),及化學(xué)效應(yīng)等生物學(xué)效應(yīng),次聲對人體各系統(tǒng)均有影響,可引起體內(nèi)細(xì)胞的生物學(xué)變化,還可對多種體外培養(yǎng)的細(xì)胞產(chǎn)生影響。次聲在腦缺血再灌注損傷等神經(jīng)系統(tǒng)疾病的防護(hù)上,有一定效應(yīng),還可影響神經(jīng)前體細(xì)胞的增殖。而BMSCs也已經(jīng)廣泛用于神經(jīng)系統(tǒng)疾病,其機(jī)制可能是通過在體內(nèi)的神經(jīng)分化實(shí)現(xiàn)的,所以開展次聲對BMSCs的生物學(xué)效應(yīng)研究就是一項(xiàng)非常有必要的課題。 目前,國內(nèi)學(xué)者多研究次聲對成骨細(xì)胞、神經(jīng)前體細(xì)胞、小角質(zhì)細(xì)胞、角膜細(xì)胞、癌細(xì)胞等細(xì)胞的超微結(jié)構(gòu)及增殖能力等方面的影響。對BMSCs的研究還鮮有報(bào)道,但是已有人做過低強(qiáng)度超聲對BMSCs所產(chǎn)生的生物學(xué)效應(yīng)的研究,發(fā)現(xiàn)低強(qiáng)度超聲可以促進(jìn)BMSCs的增殖及軟骨定向分化,次聲同樣作為一種機(jī)械波,在這方面可能會有共通之處。國外方面：近年來,國外學(xué)者對次聲在生物醫(yī)學(xué)界的研究較少,多為次聲對機(jī)體產(chǎn)生的損傷效應(yīng)。在細(xì)胞培養(yǎng)方面,大多數(shù)人認(rèn)為某些細(xì)胞因子及氨基酸對細(xì)胞的培養(yǎng)及分化有一定的作用,但也有人已經(jīng)提出力學(xué)因素在這一方面的作用。 次聲波的生物學(xué)效應(yīng)主要由頻率、時(shí)間、強(qiáng)度等參數(shù)來決定,本課題主要觀察低壓級次聲不同作用時(shí)間對BMSCs所產(chǎn)生的生物學(xué)效應(yīng)。 研究目的 觀察次聲對骨髓間充質(zhì)干細(xì)胞(Bone Marrow Stromal Stem Cells, BMSCs)的生物學(xué)效應(yīng),包括增殖、凋亡和周期分布,以及超微結(jié)構(gòu)的影響,探討次聲合理應(yīng)用的時(shí)間參數(shù)。材料與方法： 1、細(xì)胞的培養(yǎng)和鑒定：用頸椎脫位法處死SD大鼠,分離獲得股骨和脛骨,用DMEM/F12培養(yǎng)基反復(fù)沖洗骨髓腔,將沖洗后的骨髓懸液室溫狀態(tài)下離心1000r.p.m,5min。離心結(jié)束后,倒掉上清液,用含10%胎牛血清和90%DMEM/F12培養(yǎng)基的細(xì)胞全培液重懸細(xì)胞,在37℃、5%CO2孵箱中培養(yǎng),2天后換液,之后每隔2天換液1次。在原代細(xì)胞充分融合后,用含0.02%EDTA的25%的胰酶和PBS液(1：1)消化細(xì)胞后,傳代純化細(xì)胞,所有實(shí)驗(yàn)都采用p3代細(xì)胞,采用流式細(xì)胞術(shù)檢測間充質(zhì)干細(xì)胞的表面抗原標(biāo)志CD29, CD90和CD45的表達(dá)情況,用臺盼藍(lán)檢測細(xì)胞活力。 2、試驗(yàn)方法：取P3代細(xì)胞分為：實(shí)驗(yàn)組(次聲處理10min,30min,60min)和對照組(空氣暴露相同時(shí)間),細(xì)胞處理結(jié)束后,迅速轉(zhuǎn)入37℃、5%CO2孵箱中培養(yǎng)。采用CCK8法檢測細(xì)胞的增殖活性,流式細(xì)胞術(shù)進(jìn)行細(xì)胞凋亡和細(xì)胞周期分析,以及采用掃描電鏡和透射電鏡觀察細(xì)胞的超微結(jié)構(gòu)變化。 增殖實(shí)驗(yàn)在96孔板進(jìn)行,每組1板共6板。實(shí)驗(yàn)組和對照組細(xì)胞數(shù)均調(diào)整為4000個(gè)/100ul/孔,每組12孔。細(xì)胞鋪板后放于孵箱中培養(yǎng)3個(gè)小時(shí)基本貼壁后,對細(xì)胞進(jìn)行次聲和空氣暴露的處理,之后在每天的相同時(shí)間段處理細(xì)胞1次,連續(xù)處理3天,處理結(jié)束后,迅速將細(xì)胞轉(zhuǎn)入孵箱中培養(yǎng)。細(xì)胞鋪板后48小時(shí)進(jìn)行CCK8檢測,每天一次,共觀察3天,每天每培養(yǎng)板取4個(gè)復(fù)孔。 凋亡實(shí)驗(yàn)和周期分析實(shí)驗(yàn)時(shí),將細(xì)胞用培養(yǎng)基重懸后,移至5ml凍存管中進(jìn)行次聲和對照組的處理,處理結(jié)束后將細(xì)胞吹打重懸、轉(zhuǎn)入25cm2培養(yǎng)瓶中,用含10%胎牛血清的DMEM/F12培養(yǎng)基在孵箱中培養(yǎng)3天。 電鏡超微結(jié)構(gòu)觀察實(shí)驗(yàn)時(shí),將細(xì)胞用培養(yǎng)基重懸后,移至5ml凍存管中進(jìn)行次聲和對照組的處理,處理時(shí)間為60min。用于掃描電鏡觀察的細(xì)胞在處理完后,用全培液重懸并且轉(zhuǎn)入放有消毒蓋玻片的培養(yǎng)皿中,迅速移入孵箱中培養(yǎng),過夜。用于透射電鏡觀察的細(xì)胞在處理完后,轉(zhuǎn)入25cm2培養(yǎng)瓶中,用含10%胎牛血清的DMEM/F12培養(yǎng)基在孵箱中培養(yǎng)4天。 各組數(shù)據(jù)以(X±S)表示,采用Spss13.0軟件處理。對OD值的處理用析因設(shè)計(jì)資料的方差分析,對凋亡率和細(xì)胞周期分布率用t檢驗(yàn)分析,P0.05為差異有統(tǒng)計(jì)學(xué)意義。 結(jié)果 1、臺盼藍(lán)結(jié)果顯示,細(xì)胞活力在95%以上,可以進(jìn)行下一步實(shí)驗(yàn)。 2、表面標(biāo)志物檢測顯示：CD29和CD90陽性,CD45陰性。 3、采用CCK8法檢測的結(jié)果：采用析因設(shè)計(jì)的方差分析顯示,經(jīng)次聲處理10min、30min和60mmin的BMSCs,培養(yǎng)48h后,OD值分別為(0.929±0.042；1.094±0.013；1.410±0.016),培養(yǎng)72h后,OD值分別為(1.480±0.001；1.348±0.027；1.493±0.017),培養(yǎng)96h后,OD值分別為(1.774±0.127；1.731±0.062；1.833±0.054),對照組三個(gè)培養(yǎng)時(shí)間的OD值分別為(1.148±0.088；1.147±0.030；1.112±0.051),(1.479±0.051；1.267±0.006；1.227±0.126)和(1.567±0.032；1.563±0.043；1.632±0.071),可見培養(yǎng)72h后次聲組細(xì)胞OD值均大于對照組,且隨著處理時(shí)間的延長,細(xì)胞OD值呈現(xiàn)遞增或先減后增的趨勢,在各觀察點(diǎn)次聲處理60min的BMSCs OD值都最高。采用t檢驗(yàn)進(jìn)行單獨(dú)效應(yīng)分析,可見兩組間差異有統(tǒng)計(jì)學(xué)意義(P0.05)。 4、細(xì)胞凋亡結(jié)果顯示：次聲處理10mmin和60min時(shí),BMSCs的早期凋亡率分別為(1.07%±0.12%和0.97%±0.21%),相應(yīng)對照組的早期凋亡率分別為(1.43%±0.06%和3.33%±0.15%),可見次聲處理10min和60min后細(xì)胞早期凋亡率降低(P0.01),處理60min還可降低BMSCs的中晚期凋亡率、提高正常細(xì)胞百分率(P0.01),處理30min時(shí),對BMSCs的細(xì)胞凋亡沒有影響。 5、細(xì)胞周期結(jié)果顯示：次聲處理10min和30min相比,可對BMSCs的細(xì)胞周期產(chǎn)生相反的影響,次聲處理10min時(shí),靜止期細(xì)胞多于對照組(P0.05),處理30min時(shí)靜止期細(xì)胞少于對照組(P0.01),而次聲處理60min對BMSCs的細(xì)胞周期無影響(P0.05) 6、掃描電鏡下顯示：次聲處理60min培養(yǎng)1天的細(xì)胞在細(xì)胞大小、形狀、表面微絨毛程度與空氣暴露對照組有較大差異,次聲組細(xì)胞多舒展開,為長條狀,細(xì)胞較長,表面微絨毛較多；對照組含有大量圓形透亮細(xì)胞,表面微絨毛較少,提示細(xì)胞死亡或活力低下。 7、透射電鏡下顯示：次聲處理60min培養(yǎng)4天的細(xì)胞與對照組細(xì)胞相比,細(xì)胞狀態(tài)良好,細(xì)胞器大量存在,細(xì)胞核沒有明顯改變,核膜清晰；對照組有大量細(xì)胞出現(xiàn)核破裂、核溶解,可見大量溶酶體出現(xiàn),提示細(xì)胞大量死亡。 結(jié)論 1.次聲可以促進(jìn)BMSCs的增殖,沒有發(fā)現(xiàn)次聲引起B(yǎng)MSCs的凋亡,次聲處理細(xì)胞60mmin后可抑制細(xì)胞凋亡；次聲作用3天后大多數(shù)BMSCs停留在靜止期,但次聲可以干擾BMSCs的周期分布。次聲可對BMSCs的超微結(jié)構(gòu)產(chǎn)生影響,提示次聲可以改善細(xì)胞活力。 2.次聲作用60min后,可以穩(wěn)定的促進(jìn)細(xì)胞增殖,抑制細(xì)胞凋亡,不改變細(xì)胞的正常周期分布,對細(xì)胞超微結(jié)構(gòu)有積極作用,比較適合在骨髓間充質(zhì)干細(xì)胞培養(yǎng)中應(yīng)用。
[Abstract]:Research background
The research and application of stem cells is a new way and new hope for regenerative repair and treatment of major diseases. At present, the key technologies such as isolation, identification, culture, expansion, preservation and resuscitation of stem cells have been highly concerned by various research institutions and individuals, and the advantages of BMSCs because of their abundant sources are popular.
The infrasonic wave is a mechanical vibration wave in the range of 0.0001 ~ 20Hz, which may have biological effects such as mechanical effect, thermothermal effect and chemical effect. Infrasound has influence on all systems of human body, can cause biological changes of cells in the body, and can also affect a variety of cultured cells in vitro. Infrasound in cerebral ischemia reperfusion injury and so on. The protection of systemic diseases has a certain effect and can affect the proliferation of neural precursor cells. And BMSCs has also been widely used in nervous system diseases. The mechanism may be realized through the differentiation of nerve in the body, so it is a very necessary subject to carry out the study of the biological effects of infrasound on BMSCs.
At present, domestic scholars have studied the effects of infrasound on the ultrastructure and proliferation of osteoblasts, neural precursor cells, small keratinocytes, corneal cells, cancer cells and so on. There are few reports on the study of BMSCs. However, there has been a study on the biological effects of low intensity ultrasound on BMSCs. Sound can promote the proliferation of BMSCs and the directional differentiation of cartilage. The infrasound is also a mechanical wave, and there may be a common place in this area. In recent years, foreign scholars have studied the infrasound in the biomedical field less, mostly the damage effect of infrasound to the body. In the cell culture, most people think some cells. Factors and amino acids play a certain role in cell culture and differentiation, but some have suggested the role of mechanical factors in this aspect.
The biological effects of infrasonic wave are mainly determined by the parameters of frequency, time, intensity and so on. This subject is mainly to observe the biological effects of the different action time of the infrasound on the BMSCs.
research objective
To observe the biological effects of infrasound on bone marrow mesenchymal stem cells (Bone Marrow Stromal Stem Cells, BMSCs), including proliferation, apoptosis and periodic distribution, and the influence of ultrastructure, and discuss the time parameters of rational use of infrasound. Materials and methods:
1, cell culture and identification: the SD rats were killed by the cervical dislocation method, the femur and tibia were isolated and the bone marrow cavity was washed with DMEM/F12 medium. The bone marrow suspension after the rinse was centrifuged for 1000r.p.m at room temperature. After the 5min. centrifugation, the supernatant was dropped and the full culture of the cells containing 10% fetal bovine serum and 90%DMEM/F12 medium was suspended. The cells were cultured in the 5%CO2 incubator at 37 degrees C, then changed liquid after 2 days, and then changed 1 times every 2 days. After the primary cells were fully fused, the cells were purified with 0.02%EDTA 25% trypsin and PBS solution (1:1), and all the cells were purified. All the experiments were P3 cells, and the surface antigen marker CD29, CD90 and CD90 were detected by flow cytometry. The expression of CD45 was detected by trypan blue.
2, the test method: the P3 generation cells were divided into: the experimental group (infrasound treatment 10min, 30min, 60min) and the control group (air exposure for the same time). After the cell treatment ended, the cells were quickly transferred to 37 C and incubated in 5%CO2 incubator. The cell proliferation activity was detected by CCK8 method, cell apoptosis and cell cycle analysis were carried out by flow cytometry, and the scanning electricity was used. The ultrastructural changes of the cells were observed by microscope and transmission electron microscope.
The proliferation experiment was carried out in 96 orifice plates with 1 plates in each group of 6 plates. The number of cells in the experimental group and the control group were adjusted to 4000 /100ul/ holes, with 12 holes in each group. After the cell planks were placed in the incubator for 3 hours and basically adhered to the wall, the cells were treated with infrasound and air exposure, and the cells were treated for 1 times in the same time each day for 3 days. At the end of the treatment, the cells were transferred to incubator rapidly. CCK8 test was carried out 48 hours after cell planking. Once a day, 3 days were observed, and 4 duplicate holes were taken per culture plate every day.
In the apoptosis and cycle analysis experiments, the cells were suspended in the medium of suspension and transferred to the 5ml cryopreservation tube for the treatment of the infrasound and the control group. After the treatment, the cells were overhung and transferred into the 25cm2 culture bottle, and the DMEM/F12 medium containing 10% fetal bovine serum was cultured for 3 days in the incubator.
When the ultrastructure of the electron microscope was observed, the cells were suspended in the culture medium and transferred to the 5ml cryopreservation tube for the infrasound and the control group. The cells treated with the scanning electron microscope were treated with the scanning electron microscope after the treatment. The cells were suspended in full culture and transferred into the culture dish with disinfectant glass slide. The cells were quickly moved into the incubator and used for the night. The cells observed after transmission electron microscope were transferred to 25cm2 culture bottle after treatment, and incubated in DMEM/F12 incubator containing 10% fetal bovine serum for 4 days.
The data of each group were expressed by (X + S) and treated with Spss13.0 software. The variance analysis of the design data was used for the treatment of OD values. The apoptosis rate and the cell cycle distribution rate were analyzed by t test. The difference of P0.05 was statistically significant.
Result
1, trypan blue showed that the cell viability was above 95%, and the next experiment could be carried out.
2, surface marker detection showed that CD29 and CD90 were positive and CD45 negative.
3, the results of CCK8 test: the analysis of variance in the factorial design showed that after the secondary sound treatment of 10min, 30min and 60mmin BMSCs, after cultivating 48h, the OD values were respectively (0.929 + 0.042; 1.094 + 0.013; 1.410 + 0.016). After cultivating 72h, the OD values were respectively (1.480 + 0.001; 1.348 + 0.027; 1.493 + 0.017). After developing 96h, the OD values were respectively (0.042) 27, 1.731 + 0.062; 1.833 + 0.054), the three cultures of the control group were (1.148 + 0.088; 1.147 + 0.030; 1.112 + 0.051). On the other hand, the BMSCs o value of 60min was the highest at all observation points, and the difference between the two groups was statistically significant (P0.05).
4, the apoptosis results showed that the early apoptosis rate of BMSCs was (1.07% + 0.12% and 0.97% + 0.21%), respectively (1.43% + 0.06% and 3.33% + 0.15%) in the corresponding control group, respectively (1.43% + 0.06% and 3.33% + 0.15%) in the corresponding control group, and the early apoptosis rate decreased (P0.01) after the infrasound treatment of 10min and 60min (P0.01). The treatment of 60min could also reduce the middle and late BMSCs of the BMSCs. The apoptosis rate increased and the percentage of normal cells (P0.01) increased. The treatment of 30min had no effect on the apoptosis of BMSCs.
5, the cell cycle results showed that the infrasound treatment compared with 10min and 30min could have the opposite effect on the cell cycle of BMSCs. When the infrasound treatment was 10min, the resting cells were more than the control group (P0.05), and the stationary phase cells were less than the control group (P0.01) when treating 30min, while the infrasound processing 60min had no effect on the cell cycle of BMSCs (P0.05).
6, the scanning electron microscope showed that the cell size, shape, surface microvilli degree and air exposure control group were different in the cell size, shape and surface microvilli in the secondary sound treatment for 1 days. The cells in the infrasonic group were long, long and with more surface microvilli in the infrasonic group. The control group contained a large number of round bright cells and less surface microvilli, suggesting cells. Death or low vitality.
7, the transmission electron microscope showed that the cells in the secondary sound treatment for 4 days of 60min culture were in good condition, there were a large number of organelles, the nuclei had no obvious changes and the nuclear membrane was clear, and a large number of cells in the control group had nuclear rupture, nuclear dissolution, and a large number of lysosomes appeared, suggesting a large number of cells died.
conclusion
1. infrasound can promote the proliferation of BMSCs. No infrasound causes apoptosis of BMSCs. Infrasound treatment cells 60mmin can inhibit cell apoptosis. Most of BMSCs stays at rest after 3 days of infrasound action, but infrasound can interfere with the periodic distribution of BMSCs. Infrasound can affect the ultrastructure of BMSCs, suggesting that infrasound can improve cell survival. Power.
After the 2. infrasound action of 60min, it can promote cell proliferation, inhibit cell apoptosis, do not change the normal distribution of cell cycle, have positive effect on cell ultrastructure, and be suitable for the application of bone marrow mesenchymal stem cell culture.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R329

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