人角朊干细胞的免疫磁珠分选及siRNA干扰其CCL20表达的初步研究

发布时间：2018-03-17 04:37

本文选题：角朊干细胞　切入点：HaCaT细胞　出处：《第三军医大学》2007年硕士论文　论文类型：学位论文

【摘要】： 目的 1.从人包皮组织获得角朊细胞,将其进行体外无血清培养,探索其体外培养的生长特点。利于免疫磁珠细胞分选法(Magnetic Activated Cell Sorting,MACS)结合人胎盘Ⅳ型胶原粘附法从角朊细胞中分离出角朊干细胞后进行无血清培养,寻求一种理想有效的人角朊干细胞体外分离培养技术。 2.在本课题组前期已经成功构建两种人CCL20基因特异性小干扰RNA (small interference RNA,siRNA)重组慢病毒表达载体的基础上,补充测序鉴定出一种人CCL20基因特异性siRNA重组慢病毒阴性对照载体。同时,将构建的重组慢病毒载体以包装细胞293FT包装生产,最后以慢病毒颗粒感染人角朊细胞株HaCaT细胞和角朊干细胞,并初步检测siRNA对于角朊细胞表达CCL20的干扰效果。方法 1.按照Dispase酶-胰酶两步消化法从人包皮组织获得角朊细胞(Keratinocyte,KC),以人胎盘Ⅳ型胶原按照5μg/cm2的密度包被培养瓶,将角朊细胞以无血清培养基(defined keratinocyte-serum free medium,DK-SFM)进行体外原代及传代培养,并观察其生长形态变化、克隆形成,并对原代培养的KC中的KSC以细胞表面分子CD49f(整合素α6)、CD71(转铁蛋白受体)、CD29 (整合素β1)做表型检测。 2.以免疫磁珠法对KC细胞中的角朊干细胞(keratinocyte stem cells,KSC)进行分选,分析其分选的纯度和比例,再结合人胎盘Ⅳ型胶原粘附法将分选所得的细胞进行体外无血清培养,观察其生长情况及克隆形成。 3.将含有CCCL20-siRNA慢病毒载体重组质粒的大肠杆菌(escherichia coli, E.coli)的菌液以划线法接种于含有Amp 60μg/ml的LB平板上,筛选出阳性菌落、扩增后提取质粒,进行DNA测序鉴定。 4.重组慢病毒表达载体质粒pHSER-CCL20-siRNA-GFP-SIN、慢病毒包装质粒Lentipack和慢病毒包膜蛋白质粒Lentienv按照一定比例混合,与转染试剂jetPEI一同转染包装细胞293FT,24h后观察其荧光表达情况,并分别在48h、72h、96h后,收集含有慢病毒颗粒的培养基上清液,并保存在-80℃。 5.分别将慢病毒颗粒加入至培养至50-60%融合的HaCaT和人角朊干细胞,并设立空白对照组。24 h后,并在所有细胞中加入刺激因子TNF-α、IL-1β和DK-SFM的混合液,使TNF-α和IL-1β的终浓度均为100ng/ml。48h后收集细胞上清液,进行人CCL20的酶联免疫吸附测定(Enzyme linked-immuno-sorbent assay ,ELISA),在酶标仪上检测其OD450值。根据试剂盒中标准品的浓度-OD450值,求出其拟合曲线和方程,将样品OD450值代入方程,得到样品中CCL20的蛋白浓度。比较几种慢病毒颗粒和对照组的CCL20浓度,初步评价siRNA干扰对于TNF-α和IL-1β共同刺激下的人角朊细胞株HaCaT和角朊干细胞表达CCL20的下调作用。结果 1.角朊细胞以无血清培养基可在体外稳定培养35.13±7.59天,传3.87±0.83代,原代细胞克隆形成时间为5.75±0.90天。原代与传代细胞的克隆形成时间和传代时间差异不明显(P0.05),细胞经传代培养后细胞数无明显增加。 2. CD49f、CD71直接免疫荧光双重标记的原代培养角朊细胞中CD49fbriCD71dim细胞的比例为46.6±2.1%,CD29直接免疫荧光标记原代培养角朊细胞的阳性率为65.8±2.3%。 3.新鲜的人角朊细胞悬液经MACS法分选后所得的CD49fbriCD71dim细胞比率达(12.2±7.1)%。CD49fbriCD71dim细胞经培养可见细胞为典型的上皮样特征,呈铺路石样形态,高核浆比例,细胞紧密排列,轮廓清楚,折光性好,并可在5-7天左右可形成全克隆,符合角朊干细胞的特点。 4.构建的1种重组慢病毒阴性对照载体通过测序验证载体构建成功。 5.成功利用293FT细胞包装已经构建成功的重组载体质粒,生产出慢病毒颗粒,观察到其有较高的感染效应。慢病毒颗粒感染HaCaT和角朊干细胞后通过人CCL20酶联免疫吸附测定实验,初步观察到两种人CCL20基因特异性siRNA重组慢病毒表达载体对于人角朊细胞表达CCL20有一定的下调作用。结论 1.对人KC的体外无血清培养方法进行了观察,探索出了其体外无血清培养的生长特性和规律。 2.将MACS分选法与人胎盘Ⅳ型胶原粘附法相结合从皮肤组织的表皮细胞悬液中直接分离出较高比例的干细胞,在如何对KSC进行大量纯化的方法学上做了一次新的探索,为后续研究提供了新的备选技术路线。 3.补充测序验证成功了1种人CCL20基因特异性siRNA重组慢病毒阴性对照载体。 4.以TNF-α和IL-1β共同刺激感染了CCL20-siRNA慢病毒颗粒的人角朊细胞株HaCaT和人角朊干细胞,并利用ELISA实验,测定两种细胞表达CCL20的变化情况,初步观察到siRNA对其表达CCL20的有一定的下调作用,从而为下一步筛选相应的阳性克隆和进一步评价其siRNA干扰效果奠定了基础。
[Abstract]:objective
1. from human foreskin tissue obtained keratinocytes, the in vitro serum-free culture, explore the growth characteristics of the cultured in vitro for immunomagnetic cell sorting (Magnetic Activated Cell Sorting, MACS) combined with human placental collagen adhesion by separating keratinocyte stem cells from keratinocytes were cultured in serum free medium. To seek an ideal, effective human keratinocyte stem cells isolated and cultured in vitro.
2. in ourprevious has successfully constructed two CCL20 gene specific small interfering RNA (siRNA small interference RNA) expression of recombinant lentiviral vector on the basis of sequencing identified a specific CCL20 gene siRNA lentiviral vector for negative control. At the same time, the recombinant lentiviral vectors the packaging cell 293FT packaging production, and finally to the lentivirus infection of human keratinocyte cell line HaCaT cells and keratinocyte stem cells, and preliminary detection of siRNA for keratinocyte expression of CCL20 jamming effect.
Method
According to the 1. Dispase enzyme trypsin two step digestion method from human foreskin tissue obtained keratinocytes (Keratinocyte, KC), with human placenta collagen with 5 g/cm2 density coated bottles, the keratinocytes in serum-free medium (defined keratinocyte-serum free medium, DK-SFM) were primary cultured and passaged culture, and to observe the morphological changes of growth, colony formation, and on the primary culture of KC KSC on cell surface molecule CD49f (integrin alpha 6), CD71 (transferrin receptor), CD29 (integrin beta 1) do phenotype detection.
2. by immunomagnetic beads on KC cells in keratinocyte stem cells (keratinocyte stem cells, KSC) were separated, and the proportion of the separation purity analysis, combined with human placental collagen adhesion method will be separated from cells in vitro serum-free medium to observe the growth and colony formation.
3., we inoculate the Escherichia coli (E.coli) containing CCCL20-siRNA lentiviral vector recombinant plasmid with streak method on the LB plate containing Amp 60 g/ml g/ml, and select positive colonies. After amplification, we extract plasmid and identify it by DNA sequencing.
4. recombinant lentiviral expression vector plasmid pHSER-CCL20-siRNA-GFP-SIN, lentiviral packaging plasmid Lentipack and lentiviral envelope protein Lentienv in certain proportion, and the transfection reagent jetPEI to the transfected 293FT cell after 24h to observe the expression of fluorescence, and respectively in 48h, 72h, 96h, medium supernatant collection containing lentiviral particles, and stored in -80 C.
5. the lentivirus particles added to the culture to the fusion of 50-60% HaCaT and human keratinocyte stem cells, and established the control group.24 after h, and join in all cell stimulating factor TNF- alpha, IL-1 beta and DK-SFM mixture, the final concentration of TNF- alpha and IL-1 beta were used in cell supernatant 100ng/ml.48h, human CCL20 ELISA (Enzyme linked-immuno-sorbent, assay, ELISA) on the enzyme labelling instrument to detect the value of OD450. According to the concentration of standard -OD450 kit in value, calculate the fitting curve and the equation, the sample OD450 value into the equation, get the protein concentration in the samples CCL20. Comparison of several concentrations of CCL20 lentivirus and the control group, the preliminary evaluation of siRNA interference for TNF- alpha and IL-1 beta together under the stimulation of human keratinocyte cell line HaCaT and keratinocyte stem cells CCL20 expression down-regulation.
Result
1. keratinocytes in serum-free medium in vitro cultured 35.13 + 7.59 days, 3.87 + 0.83, primary cell clone formation time was 5.75 + 0.90 days. The primary cloning and cell formation time and time were no significant difference (P0.05), cells were cultured cell number increased significantly.
2. CD49f, CD71 direct immunofluorescence double labeled CD49fbriCD71dim cells in primary cultured keratinocytes were 46.6 + 2.1%, and the positive rate of CD29 immunofluorescence labeling primary cultured keratinocytes was 65.8 + 2.3%..
The ratio of CD49fbriCD71dim cells 3. fresh human keratinocyte cell suspension by MACS method after the separation of (12.2 + 7.1)%.CD49fbriCD71dim cells cultured cells as the typical characteristics of epithelioid, cobblestone morphology, high karyoplasmic ratio, cells arranged tightly, clear outline and good refraction, and can form the clones in 5-7 days, with stem cell characteristics of keratin.
4. the 1 recombinant lentivirus negative control vectors were constructed successfully by sequencing.
293FT cell packing has been successfully used to construct the recombinant plasmid successfully produced 5., lentivirus, observed its high infection effect. Lentivirus infected HaCaT and keratinocyte stem cells by human CCL20 ELISA experiments, preliminary observation to two specific human CCL20 gene siRNA recombinant lentivirus the expression vector for human keratinocytes expressing CCL20 could down regulation.
conclusion
1. the serum-free culture of KC in vitro was observed, and the growth characteristics and regularity of the serum-free culture in vitro were explored.
2. the MACS sorting method and human placenta collagen adhesion method combining with skin epidermal cell suspension in the isolated high proportion of stem cells, made a new exploration in how to carry out the method for purification of a large number of KSC, provides a new alternative technical route for further study.
3. supplemental sequencing proved that 1 human CCL20 gene specific siRNA recombinant lentivirus negative control vectors were successfully used.
4. to TNF- alpha and IL-1 beta stimulation were infected with CCL20-siRNA lentivirus human keratinocyte cell line HaCaT and human keratinocyte stem cells, and using the ELISA test, the determination of two kinds of cells expressing CCL20 changes to CCL20 siRNA, preliminary observation on its expression has certain effect to cut, for the next step the positive clones were screened and further evaluate its corresponding siRNA interference effect of the foundation.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329.2

【参考文献】