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  本文選題：角朊干細胞　切入點：HaCaT細胞　出處：《第三軍醫(yī)大學》2007年碩士論文　論文類型：學位論文

【摘要】： 目的 1.從人包皮組織獲得角朊細胞,將其進行體外無血清培養(yǎng),探索其體外培養(yǎng)的生長特點。利于免疫磁珠細胞分選法(Magnetic Activated Cell Sorting,MACS)結合人胎盤Ⅳ型膠原粘附法從角朊細胞中分離出角朊干細胞后進行無血清培養(yǎng),尋求一種理想有效的人角朊干細胞體外分離培養(yǎng)技術。 2.在本課題組前期已經成功構建兩種人CCL20基因特異性小干擾RNA (small interference RNA,siRNA)重組慢病毒表達載體的基礎上,補充測序鑒定出一種人CCL20基因特異性siRNA重組慢病毒陰性對照載體。同時,將構建的重組慢病毒載體以包裝細胞293FT包裝生產,最后以慢病毒顆粒感染人角朊細胞株HaCaT細胞和角朊干細胞,并初步檢測siRNA對于角朊細胞表達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,將角朊細胞以無血清培養(yǎng)基(defined keratinocyte-serum free medium,DK-SFM)進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,并對原代培養(yǎng)的KC中的KSC以細胞表面分子CD49f(整合素α6)、CD71(轉鐵蛋白受體)、CD29 (整合素β1)做表型檢測。 2.以免疫磁珠法對KC細胞中的角朊干細胞(keratinocyte stem cells,KSC)進行分選,分析其分選的純度和比例,再結合人胎盤Ⅳ型膠原粘附法將分選所得的細胞進行體外無血清培養(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、擴增后提取質粒,進行DNA測序鑒定。 4.重組慢病毒表達載體質粒pHSER-CCL20-siRNA-GFP-SIN、慢病毒包裝質粒Lentipack和慢病毒包膜蛋白質粒Lentienv按照一定比例混合,與轉染試劑jetPEI一同轉染包裝細胞293FT,24h后觀察其熒光表達情況,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和人角朊干細胞,并設立空白對照組。24 h后,并在所有細胞中加入刺激因子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后收集細胞上清液,進行人CCL20的酶聯免疫吸附測定(Enzyme linked-immuno-sorbent assay ,ELISA),在酶標儀上檢測其OD450值。根據試劑盒中標準品的濃度-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照組的CCL20濃度,初步評價siRNA干擾對于TNF-α和IL-1β共同刺激下的人角朊細胞株HaCaT和角朊干細胞表達CCL20的下調作用。 結果 1.角朊細胞以無血清培養(yǎng)基可在體外穩(wěn)定培養(yǎng)35.13±7.59天,傳3.87±0.83代,原代細胞克隆形成時間為5.75±0.90天。原代與傳代細胞的克隆形成時間和傳代時間差異不明顯(P0.05),細胞經傳代培養(yǎng)后細胞數無明顯增加。 2. CD49f、CD71直接免疫熒光雙重標記的原代培養(yǎng)角朊細胞中CD49fbriCD71dim細胞的比例為46.6±2.1%,CD29直接免疫熒光標記原代培養(yǎng)角朊細胞的陽性率為65.8±2.3%。 3.新鮮的人角朊細胞懸液經MACS法分選后所得的CD49fbriCD71dim細胞比率達(12.2±7.1)%。CD49fbriCD71dim細胞經培養(yǎng)可見細胞為典型的上皮樣特征,呈鋪路石樣形態(tài),高核漿比例,細胞緊密排列,輪廓清楚,折光性好,并可在5-7天左右可形成全克隆,符合角朊干細胞的特點。 4.構建的1種重組慢病毒陰性對照載體通過測序驗證載體構建成功。 5.成功利用293FT細胞包裝已經構建成功的重組載體質粒,生產出慢病毒顆粒,觀察到其有較高的感染效應。慢病毒顆粒感染HaCaT和角朊干細胞后通過人CCL20酶聯免疫吸附測定實驗,初步觀察到兩種人CCL20基因特異性siRNA重組慢病毒表達載體對于人角朊細胞表達CCL20有一定的下調作用。 結論 1.對人KC的體外無血清培養(yǎng)方法進行了觀察,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型膠原粘附法相結合從皮膚組織的表皮細胞懸液中直接分離出較高比例的干細胞,在如何對KSC進行大量純化的方法學上做了一次新的探索,為后續(xù)研究提供了新的備選技術路線。 3.補充測序驗證成功了1種人CCL20基因特異性siRNA重組慢病毒陰性對照載體。 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胞株HaCaT和人角朊干細胞,并利用ELISA實驗,測定兩種細胞表達CCL20的變化情況,初步觀察到siRNA對其表達CCL20的有一定的下調作用,從而為下一步篩選相應的陽性克隆和進一步評價其siRNA干擾效果奠定了基礎。
[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.

【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R329.2

【參考文獻】

相關期刊論文 前3條

1 董征學;彭代智;劉敬;周新;田易;李芳;嚴泉;林恒;王勇;周光前;;人CCL20基因特異性shRNA重組慢病毒表達載體的構建及測序[J];第三軍醫(yī)大學學報;2006年10期

2 胡大海,陳璧,湯朝武,徐明達,賈赤宇;pcDNA_3-hbFGF轉染角朊細胞對真皮成纖維細胞增殖的生物學效應[J];第四軍醫(yī)大學學報;1999年05期

3 岳海嶺,彭代智;CCL20的結構與功能[J];免疫學雜志;2004年S1期

，

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