本文選題：角朊干細(xì)胞　切入點(diǎn)：HaCaT細(xì)胞　出處：《第三軍醫(yī)大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的 1.從人包皮組織獲得角朊細(xì)胞,將其進(jìn)行體外無血清培養(yǎng),探索其體外培養(yǎng)的生長特點(diǎn)。利于免疫磁珠細(xì)胞分選法(Magnetic Activated Cell Sorting,MACS)結(jié)合人胎盤Ⅳ型膠原粘附法從角朊細(xì)胞中分離出角朊干細(xì)胞后進(jìn)行無血清培養(yǎng),尋求一種理想有效的人角朊干細(xì)胞體外分離培養(yǎng)技術(shù)。 2.在本課題組前期已經(jīng)成功構(gòu)建兩種人CCL20基因特異性小干擾RNA (small interference RNA,siRNA)重組慢病毒表達(dá)載體的基礎(chǔ)上,補(bǔ)充測(cè)序鑒定出一種人CCL20基因特異性siRNA重組慢病毒陰性對(duì)照載體。同時(shí),將構(gòu)建的重組慢病毒載體以包裝細(xì)胞293FT包裝生產(chǎn),最后以慢病毒顆粒感染人角朊細(xì)胞株HaCaT細(xì)胞和角朊干細(xì)胞,并初步檢測(cè)siRNA對(duì)于角朊細(xì)胞表達(dá)CCL20的干擾效果。 方法 1.按照Dispase酶-胰酶兩步消化法從人包皮組織獲得角朊細(xì)胞(Keratinocyte,KC),以人胎盤Ⅳ型膠原按照5μg/cm2的密度包被培養(yǎng)瓶,將角朊細(xì)胞以無血清培養(yǎng)基(defined keratinocyte-serum free medium,DK-SFM)進(jìn)行體外原代及傳代培養(yǎng),并觀察其生長形態(tài)變化、克隆形成,并對(duì)原代培養(yǎng)的KC中的KSC以細(xì)胞表面分子CD49f(整合素α6)、CD71(轉(zhuǎn)鐵蛋白受體)、CD29 (整合素β1)做表型檢測(cè)。 2.以免疫磁珠法對(duì)KC細(xì)胞中的角朊干細(xì)胞(keratinocyte stem cells,KSC)進(jìn)行分選,分析其分選的純度和比例,再結(jié)合人胎盤Ⅳ型膠原粘附法將分選所得的細(xì)胞進(jìn)行體外無血清培養(yǎng),觀察其生長情況及克隆形成。 3.將含有CCCL20-siRNA慢病毒載體重組質(zhì)粒的大腸桿菌(escherichia coli, E.coli)的菌液以劃線法接種于含有Amp 60μg/ml的LB平板上,篩選出陽性菌落、擴(kuò)增后提取質(zhì)粒,進(jìn)行DNA測(cè)序鑒定。 4.重組慢病毒表達(dá)載體質(zhì)粒pHSER-CCL20-siRNA-GFP-SIN、慢病毒包裝質(zhì)粒Lentipack和慢病毒包膜蛋白質(zhì)粒Lentienv按照一定比例混合,與轉(zhuǎn)染試劑jetPEI一同轉(zhuǎn)染包裝細(xì)胞293FT,24h后觀察其熒光表達(dá)情況,并分別在48h、72h、96h后,收集含有慢病毒顆粒的培養(yǎng)基上清液,并保存在-80℃。 5.分別將慢病毒顆粒加入至培養(yǎng)至50-60%融合的HaCaT和人角朊干細(xì)胞,并設(shè)立空白對(duì)照組。24 h后,并在所有細(xì)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的終濃度均為100ng/ml。48h后收集細(xì)胞上清液,進(jìn)行人CCL20的酶聯(lián)免疫吸附測(cè)定(Enzyme linked-immuno-sorbent assay ,ELISA),在酶標(biāo)儀上檢測(cè)其OD450值。根據(jù)試劑盒中標(biāo)準(zhǔn)品的濃度-OD450值,求出其擬合曲線和方程,將樣品OD450值代入方程,得到樣品中CCL20的蛋白濃度。比較幾種慢病毒顆粒和對(duì)照組的CCL20濃度,初步評(píng)價(jià)siRNA干擾對(duì)于TNF-α和IL-1β共同刺激下的人角朊細(xì)胞株HaCaT和角朊干細(xì)胞表達(dá)CCL20的下調(diào)作用。 結(jié)果 1.角朊細(xì)胞以無血清培養(yǎng)基可在體外穩(wěn)定培養(yǎng)35.13±7.59天,傳3.87±0.83代,原代細(xì)胞克隆形成時(shí)間為5.75±0.90天。原代與傳代細(xì)胞的克隆形成時(shí)間和傳代時(shí)間差異不明顯(P0.05),細(xì)胞經(jīng)傳代培養(yǎng)后細(xì)胞數(shù)無明顯增加。 2. CD49f、CD71直接免疫熒光雙重標(biāo)記的原代培養(yǎng)角朊細(xì)胞中CD49fbriCD71dim細(xì)胞的比例為46.6±2.1%,CD29直接免疫熒光標(biāo)記原代培養(yǎng)角朊細(xì)胞的陽性率為65.8±2.3%。 3.新鮮的人角朊細(xì)胞懸液經(jīng)MACS法分選后所得的CD49fbriCD71dim細(xì)胞比率達(dá)(12.2±7.1)%。CD49fbriCD71dim細(xì)胞經(jīng)培養(yǎng)可見細(xì)胞為典型的上皮樣特征,呈鋪路石樣形態(tài),高核漿比例,細(xì)胞緊密排列,輪廓清楚,折光性好,并可在5-7天左右可形成全克隆,符合角朊干細(xì)胞的特點(diǎn)。 4.構(gòu)建的1種重組慢病毒陰性對(duì)照載體通過測(cè)序驗(yàn)證載體構(gòu)建成功。 5.成功利用293FT細(xì)胞包裝已經(jīng)構(gòu)建成功的重組載體質(zhì)粒,生產(chǎn)出慢病毒顆粒,觀察到其有較高的感染效應(yīng)。慢病毒顆粒感染HaCaT和角朊干細(xì)胞后通過人CCL20酶聯(lián)免疫吸附測(cè)定實(shí)驗(yàn),初步觀察到兩種人CCL20基因特異性siRNA重組慢病毒表達(dá)載體對(duì)于人角朊細(xì)胞表達(dá)CCL20有一定的下調(diào)作用。 結(jié)論 1.對(duì)人KC的體外無血清培養(yǎng)方法進(jìn)行了觀察,探索出了其體外無血清培養(yǎng)的生長特性和規(guī)律。 2.將MACS分選法與人胎盤Ⅳ型膠原粘附法相結(jié)合從皮膚組織的表皮細(xì)胞懸液中直接分離出較高比例的干細(xì)胞,在如何對(duì)KSC進(jìn)行大量純化的方法學(xué)上做了一次新的探索,為后續(xù)研究提供了新的備選技術(shù)路線。 3.補(bǔ)充測(cè)序驗(yàn)證成功了1種人CCL20基因特異性siRNA重組慢病毒陰性對(duì)照載體。 4.以TNF-α和IL-1β共同刺激感染了CCL20-siRNA慢病毒顆粒的人角朊細(xì)胞株HaCaT和人角朊干細(xì)胞,并利用ELISA實(shí)驗(yàn),測(cè)定兩種細(xì)胞表達(dá)CCL20的變化情況,初步觀察到siRNA對(duì)其表達(dá)CCL20的有一定的下調(diào)作用,從而為下一步篩選相應(yīng)的陽性克隆和進(jìn)一步評(píng)價(jià)其siRNA干擾效果奠定了基礎(chǔ)。
[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.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R329.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 董征學(xué);彭代智;劉敬;周新;田易;李芳;嚴(yán)泉;林恒;王勇;周光前;;人CCL20基因特異性shRNA重組慢病毒表達(dá)載體的構(gòu)建及測(cè)序[J];第三軍醫(yī)大學(xué)學(xué)報(bào);2006年10期

2 胡大海,陳璧,湯朝武,徐明達(dá),賈赤宇;pcDNA_3-hbFGF轉(zhuǎn)染角朊細(xì)胞對(duì)真皮成纖維細(xì)胞增殖的生物學(xué)效應(yīng)[J];第四軍醫(yī)大學(xué)學(xué)報(bào);1999年05期

3 岳海嶺,彭代智;CCL20的結(jié)構(gòu)與功能[J];免疫學(xué)雜志;2004年S1期

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