哺乳动物工程细胞悬浮适应—可贴壁亚群（HEK293ar）的获得及其抗失巢凋亡机制研究
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摘要
目的:
通过建立导致HEK293细胞脱离培养时完全“失巢”的培养模型,首先通过包括电镜观察、TUNEL染色、流式检测、Hoechst33342-PI双染色等多种方法研究HEK293细胞在失巢培养条件下发生的凋亡现象;并经过悬浮和贴壁培养的多轮循环,获得一种具有悬浮适应-可贴壁特性、并且能够抵御失巢凋亡的HEK293ar亚群,进而通过多种方法对该细胞亚群的失巢凋亡抗性特性进行研究,并研究该亚群是否是通过建立细胞间连接形成细胞团进而赋予细胞失巢凋亡的抗性。其次,基于失巢凋亡抗性亚群具有的失巢凋亡抗性而以细胞团形式生存和可贴壁培养的双重特点,对其可能在建立新型瞬时转染系统生产蛋白的可行性和无载体固定化细胞团培养的增殖特性进行研究。再次,以在失巢条件下可形成细胞团而生存的失巢凋亡抗性亚群HEK293ar和失巢敏感的亲本细胞HEK293这一对自然细胞模型研究HAb18G/CD147黏附分子在失巢凋亡抗性细胞中的表达差异,并研究其调节细胞间连接的作用,进而明确HAb18G/CD147调节的细胞间连接在失巢凋亡抗性调节中的作用;同时还研究另外一种黏附分子E-cadherin是否也参与细胞失巢凋亡抗性的调节,并研究这两个黏附分子在失巢凋亡抗性调节中的可能相互联系,进而研究HAb18G/CD147调节的细胞连接在抑制失巢凋亡信号调节通路中的作用和信号调节网络。最后,基于失巢条件下,细胞形态和骨架发生的显著变化,研究失巢敏感HEK293细胞和抗性亚群HEK293ar细胞在失巢条件下主要骨架蛋白的重排方式的差异,并研究HAb18G/CD147和骨架蛋白组织分子plectin之间可能的相互作用,进而研究HAb18G/CD147是否通过plectin而调节骨架重排的可能性。
方法:根据研究目的,实验内容共分为四个部分,实验操作方法简述如下。
1哺乳动物工程细胞可贴壁失巢凋亡抗性亚群(HEK293ar)的获得建立完全失巢培养模型的培养条件,通过电镜观察等研究该细胞在失巢条件下由于发生失巢凋亡而死亡的时间规律;通过有限稀释法获得HEK293的单细胞克隆株,分别研究其在失巢条件下的失巢凋亡敏感情况;研究细胞失巢模型培养和细胞贴壁培养长时间循环培养对单克隆细胞群失巢凋亡抗性的影响,进而通过细胞失巢培养和贴壁培养的多轮循环以获得具有失巢凋亡抗性的可贴壁细胞亚群;长时间失巢培养观察失巢凋亡抗性亚群的细胞形态变化,以及不同时间取样检测其发生的失巢凋亡,以验证其具有失巢凋亡抗性的特性是否稳定存在;直接将失巢培养的细胞群在贴壁条件下培养,观察细胞是否还具有重新贴壁培养的特性。最终以获得一种具有以建立细胞间连接而成团具有失巢凋亡抗性的可贴壁性细胞亚群。
2、抗性细胞亚群HEK293ar细胞在瞬时转染法生产蛋白和无载体固定化方面的应用研究
通过Lipofectin2000试剂分别在失巢敏感细胞HEK293和抗性细胞HEK293ar中高效转染pEGFP/N1,悬浮培养研究其在悬浮培养条件下生产蛋白的差异;通过Lipofectin2000试剂在抗性细胞HEK293ar中转染pEGFP/N1,连续悬浮培养并每天取样研究其在悬浮条件下生产蛋白效率的变化。重要的,通过建立高效聚乙烯亚胺转(PEI)染技术,建立基于HEK293ar细胞亚群的高效PEI瞬时转染技术策略。
3、HAb18G/CD147黏附分子参与调节细胞连接和失巢凋亡抗性的研究
以获得的失巢凋亡抗性亚群HEK293ar和亲本细胞HEK293这对细胞为模型,通过流式细胞术测定和western blot等方法研究HAb18G/CD147黏附分子的表达差异,并研究其在悬浮培养抗性细胞时形成的细胞团中的亚细胞定位;通过RNAi技术下调HAb18G/CD147表达研究其对细胞间连接和细胞凋亡抗性的影响;研究参与调节细胞间连接和凋亡抗性的另外一种黏附分子E-cadherin在这对细胞间的表达差异,并通过RNAi技术研究下调此蛋白表达对细胞间连接和细胞存活的影响;通过western blot和免疫荧光法研究下调HAb18G/CD147对E-cadherin表达和细胞生存的影响;通过额外添加EDTA和抗-E-cadherin抗体阻断细胞间连接研究封闭或破坏E-cadherin作用对细胞表达HAb18G/CD147的影响,并通过RNAi技术下调E-cadherin表达研究其对HAb18G/CD147表达的影响。通过研究添加LY294002(PI-3K通路信号竞争性抑制剂)和PD98059(ERK通路抑制剂)等信号通路抑制剂对失巢凋亡抗性亚群细胞增殖和细胞间连接的影响,研究其参与失巢凋亡抗性调节的细胞主要信号通路。
4、失巢凋亡抗性亚群在失巢培养下的细胞骨架特征和黏附分子HAb18G/CD147之间的关系研究
通过形态观察研究抗性细胞亚群HEK293ar细胞在失巢条件的细胞连接和细胞成团现象;研究抗性细胞在失巢前后的微纤维丝细胞骨架的特征;通过免疫荧光法研究HEK293ar细胞在失巢培养下actin (G-或F-)、α-tubulin和plectin的骨架重排特征以及HAb18G/CD147的亚细胞定位;采用RNAi干涉
HAb18G/CD147表达研究其对F-actin骨架重排的可能影响;通过免疫荧光法研究失巢凋亡时HAb18G/CD147和plectin参与失巢凋亡调节的现象;通过免疫荧光双标记法研究HAb18G/CD147和plectin在失巢培养抗性亚群时的相互作用,通过RNAi下调HAb18G/CD147研究其对失巢培养时这两个分子相互作用的可能影响。
结果:
1、成功建立了针对失巢培养HEK293细胞的“完全失巢”模型,并成功用于研究工程细胞HEK293在悬浮条件下发生的失巢凋亡。观察到HEK293细胞在失巢条件下发生染色体断裂、膜发泡、DNA断裂、凋亡小体形成和亚二倍体峰等典型的细胞凋亡现象。得到八株HEK293细胞单克隆细胞,研究得知,亲本细胞是由一群有不同失巢敏感程度的细胞亚群而组成的。长时间失巢培养有助于提高细胞的抗失巢凋亡能力。通过利用细胞失巢培养和贴壁培养循环获得了一种具有失巢凋亡抗性以细胞团方式生长的可贴壁细胞亚群。该亚群具有稳定的失巢凋亡抗性和重新贴壁培养的的特性。
2、贴壁转染EGFP基因后,抗性细胞HEK293ar在悬浮条件下培养到第三天时仍然维持着高效生产蛋白的能力,而亲本细胞HEK293则由于发生失巢凋亡蛋白产率和HEK293ar相比有明显下降。长时间悬浮培养HEK293ar时,可以维持初始时的高效生产蛋白的能力。利用HEK293ar细胞进行无载体固定化培养时,维持细胞活性在80-87 %,比生长速率达到0.05-0.06 hr-1,此细胞亚群可以用于无载体固定化培养。建立了基于HEK293ar细胞亚群的高效PEI转染策略,其转染效率高达65-75 %,而且在悬浮状态时可以维持高效生产蛋白的能力。
3、利用失巢凋亡抗性细胞HEK293ar和HEK293细胞这对自然细胞研究模型,证实黏附分子HAb18G/CD147内源性表达水平的提高是抗性细胞具有失巢凋亡抗性的重要原因。下调HAb18G/CD147可以导致细胞失巢培养条件下细胞间连接断裂以及细胞发生失巢凋亡。成功利用此模型研究证实E-cadherin黏附分子也参与了抗性细胞的抗性调节。下调HAb18G/CD147可以导致E-cadherin在24 hr后逐步降解,细胞间连接断裂。利用EDTA溶液以及抗E-cadherin抗体阻断细胞间连接对HAb18G/CD147表达没有显著影响,干涉E-cadherin也对HAb18G/CD147表达没有明显影响。用PI-3K通路信号竞争性抑制剂LY294002处理抗性细胞对抗性细胞的增殖和细胞间连接有影响,而用ERK通路抑制剂PD98059处理却没有同样的效果。
4、阐明了HEK293ar在失巢培养条件下细胞间连接加强和微纤维丝骨架重排的现象;抗性细胞亚群HEK293ar骨架重排在失巢条件下和贴壁下相比呈现紊乱的F-actin排布形式( aberrant cytoskeleton distribution );通过免疫荧光法研究得知,HEK293ar细胞在失巢下细胞骨架蛋白actin、α-tubulin和plectin均呈现紊乱的重排形式,而且和亲本细胞相比,其表达均有所上调。下调HAb18G/CD147导致抗性细胞HEK293ar失巢条件下的F-actin发生重排,表达变弱,呈现典型的细胞发生凋亡时的骨架重排现象,在细胞边缘以及表面突起处部分表达变强;发现抗性细胞亚群在失巢培养时,plectin和HAb18G/CD147的表达存在部分共定位,两分子在抗性细胞中的表达呈正相关(0 结论:
1、成功获得具有失巢凋亡抗性的贴壁性HEK293ar细胞亚群,以用做研究细胞间连接和工程细胞团生长时的细胞模型,此细胞亚群的获得为建立高效便宜的瞬时转染系统奠定了细胞基础,也是进行无载体固定化培养的适宜细胞亚群。
2、成功建立了基于抗性细胞亚群HEK293ar的双阶段瞬时高效转染生产药物蛋白策略,证明了此细胞亚群适宜于用于建立高效转染细胞生产蛋白平台,并且也适宜作为一种重要的无载体固定化培养的细胞亚群。
3、利用此模型细胞研究了HAb18G/CD147黏附分子调节的细胞间连接抑制失巢凋亡抗性的信号通路,得出结论为:HAb18G/CD147调节的细胞连接是以E-cadherin-PI-3K依赖的信号通路调节失巢凋亡抑制的。
4、利用此模型研究了失巢抗性细胞在失巢下的细胞骨架重排特征,证实了失巢凋亡抗性细胞在失巢下过表达骨架蛋白的特性;HAb18G/CD147可能是通过骨架蛋白的组织者plectin调节细胞骨架的重排。
Aim:
First, to establish a cell detachment model for studying anoikis of HEK293 cell. And then to acquire an anoikis-resistant subpopulation of HEK293 and study the anoikis resistance by many methods. Second, based on the acquired subpopulation, to study the advantage and feasibility of this subpopulation in transient gene expression production of protein; and then to study the feasibility and proliferation of this subpopulation using as a carrier-free immobilization culture model. Third,using the pair of HEK293 and the acquired anoikis-resistant subpopulation, to study expression change of HAb18G/CD147,an adherence molecule, in the acquired subpopulation, and then explore the function of HAb18G/CD147-mediated cell-cell contact in the anoikis-resistance regulation; And then to explore the other adherence molecule, E-caherin, in the cell-cell contact and anoikis-resistance regulation. And, to knock-down the two molecule expression using RNAi respectively and further investigate their possible correlation in the anoikis-resistance regulation. Then, with signal transduction inhibitors to treat the anoikis-resistant subpopulation and explore the possible signal pathway of anoikis resistance regulation. Lastly, based on the morphology and cytoskeleton change of cell upon suspension, to explore the cytoskeletal rearrangement of suspended cells; And then to study the possible correlation of HAb18G/CD147 and plectin in the anoikis regulation; And then to down-regulate HAb18G/CD147 expression with RNAi and study the this effect on the plectin rearrangement in the anoikis-resistant subpopulation.
Methods: All the studies can be divided into four parts according to experiments’aims. The protocols were briefly described as the following:
1、To acquire an adhesive and anoikis-resistant subpopulation To establish the“homeless”cell-detachment culture model and study the anoikis of HEK293 cell upon suspension using many methods including TUNEL assay, ultrastructure assay by TEM and SEM , flow cytometer and Hoechst33342-PI staining. And then to explore the change of cell number resulting from the anoikis upon suspension using the Hoechst33342-PI staining. And, to acquire several single cell clones from parental HEK293 cell using limited dilution cloning and then to study sensitivity of these subpopulation to cell detachment; Then to evaluate the effect of long-term cell detachment culture on the anoikis resistance of single cell clone subpopulations; To acquire the anoikis-resistant and adhesive subpopulation of HEK293 cell with sequential cycles of adherent and suspended culture. And then to study the maintaining of anoikis resistance and possibility of re-adhesive culture.
2、To study the application of anoikis-resistant cell subpopulation in transient gene expression production of protein and carrier-free immobilization culture
To transfect the pEGFP/N1 in anoikis-resistant or -sensitive HEK293 cell under adhesive couture condition and then study the efficiency of these two cell subpopulation to produce protein under suspension condition. And to transfect the pEGFP/N1 in anoikis-resistant subpopulation using Lipofectin2000 and explore the production efficiency of anoikis-resistant HEK293 cell upon suspension. Secondly, to establish the PEI transfection strategy. Thirdly, to cultivate the anoikis-resistant subpopulation of HEK293 cell in suspension and study the feasibility and proliferation of this subpopulation using as a carrier-free immobilization culture model.
3、To study whether HAb18G/CD147-mediated cell-cell contacts involve in the anoikis-regulation
Using the acquired anoikis-resistant and -sensitive HEK293 cell as cell model, to study the expression change of HAb18G/CD147 using western blot and flow cytometer between these two HEK293 subpopulation and investigate HAb18G/CD147 subcellular localization in the anoikis-resistant subpopulation using immunofluorescence staining. And then to study the effect of down-regulation of HAb18G/CD147 on the cell-cell contacts and cell survival. To study the down-regulation of HAb18G/CD147 on the E-cadherin expression using immunofluorescence staining and western blot; Then to explore the effect of inhibition of cell-cell inhibition by EDTA-treatment and an additional blockade of E-cadherin binding by an anti-E-cadherin antibody inhibited the cell-cell contacts formation on the expression of HAb18G/CD147 and cell-cell contact formation; To knock-down the E-cadherin expression by RNAi and further explore the effect of down-regulation of E-cadherin on the HAb18G/CD147 expression and cell survival. Lastly, to treat cell culture with signal transduction inhibitors and then study whether the anoikis suppression of HAb18G/CD147–mediated cell-cell contacts might be mediated through either the Ras-ERK1/2 or PI3-K-Akt cascade.
4、To study the cytoskeleton rearrangement of anoikis-resistant subpopulation upon suspension and the correlation of HAb18G/CD147 and plectin
To study the reinforcement of cell-cell contacts and cell aggregates of anoikis resistant subpopulation using morphology detection; and then to explore the F-actin distribution in anoikis-resistant cell upon suspension and adhesion culture. To study the cytoskeleton rearrangement including actin (G-or F-)、α-tubulin and plectin and HAb18G/CD147 expression of anoikis-resistant subpopulation upon suspension using a laser scanning confocal microscope; And to explore the co-localization of plectin and HAb18G/CD147 expression using double immunofluorescence staining, and to down-regulate the HAb18G/CD147 expression and evaluate this effect on the co-localization or interaction of plectin and HAb18G/CD147 expression in cytoskeleton rearrangement during anoikis suppression.
Results:
1、A cell detachment culture model adapting to HEK293 cell was successfully established. Thus, the anoikis of HEK293 cell upon suspension was studied using several methods. In detached HEK293 cell, anoikis was confirmed using TUNEL assay and ultrastructure analysis of SEM and TEM; Fragmented DNA, cell shrinkage and dead cell was confirmed to be shown in suspended HEK293 cell; Additionally, HEK293 cell was confirmed to undergo anoikis upon suspension in a time-dependent manner. Using limited dilution cloning, eight single cell clones from parental HEK293 cell were acquired successfully and were shown to be sensitive to cell detachment differently. And then the long-term cell-detachment culture was shown to have positive effect on the anoikis-resistance of cell subpopulation. Lastly, an anoikis-resistant subpopulation of HEK293 cell ( named HEK293ar ) was acquired successfully with sequential cycles of adherent and suspended culture.HEK293ar was shown to form cell-cell contacts and resist to anoikis with respect to HEK293.The acquired HEK293ar cell was shown to form cell spheroids and grow again under adhesion condition.
2、The HEK293ar cell was shown to maintain high efficiency of protein production under suspension with respect to the parental cell. In addition, even 3 days post-trasfection,the cells still maintained high production potency. Importantly,based on the HEK293ar cells, a high-efficiency PEI-transfection strategy was established; The transfection efficiency was up to 65-75 % and a dual phase transient expression for protein production in HEK293ar cells was established successfully: transfection under adhesion condition and protein production in suspension. The HEK293ar cell was shown to grow as cell spheroids and the cell viability as cell spheroids in suspension culture had little change during carrier-free immobilization culture. The specific growth rate was 0.05-0.06 hr-1.
3、The endogenetic HAb18G/CD147 expression ,an adhesive molecule, was shown to be elevated in suspended HEK293ar compared to the parental cell. And HAb18G/CD147 expression was shown to be localized in the cell-cell contacts of HEK293ar cell using immunofluorescence staining; the down-regulation of HAb18G/CD147 expression resulted in the disruption of cell-cell contacts and cell death of HEK293ar cell in suspension. In addition, E-cadherin, a Ca-dependent adhesive molecule, was also shown to be elevated in suspended HEK293ar cell, and the knock-down of E-cadherin also resulted in the inhibition of cell-cell contacts and cell death of HEK293ar cell under suspension. The down-regulation of HAb18G/CD147 was shown to result in the degradation of E-cadherin and disruption of cell-cell contacts. However, inhibition of cell-cell contacts and down-regulation of E-cadherin had no such effect on the HAb18G/CD147 expression. LY294002μmol/L decreased cellular DNA content in cell spheroids in a dose-dependent manner assayed by the cell proliferation analysis kit (Newman-Keuls Multiple Comparison Test, 50 vs. 20, P < 0.001), whereas up to 50μmol/L ERK inhibitor, PD98059 had virtually no effect. A dose-response curve of LY294002 treatment versus cell proliferation confirmed the LY294002 effect over a wide concentration range in these spheroids. Moreover, cell-cell contact formation was dramatically inhibited by LY294002 but not PD98059.
4、The reinforcement of cell adhesion and cytoskeleton rearrangement were observed in suspended HEK293ar cell. The adherent cells showed elaborated microfilaments, and appeared flattened to the dish in light microscopy; however, the suspense cells showed a rounded morphology with a diffuse distribution of F-actin throughout the cytoplasm, appearing spherical and conglobate.The down-regulation of HAb18G/CD147 was shown to induce cell to undergo anoikis and F-actin rearrangement. The reorganization of plectin seemed to be consistent with features of apoptosis; weak staining of plectin concentrated at the site of apoptotic body’s formation might suggest importance of these proteins for this process. Additionally, apoptotic cells showed a different staining pattern with CD147 included in the apoptotic bodies during anoikis. Using F-actin microfilaments as a paradigm of cytoskeleton, we observed that the interference of CD147 caused changes in the organization of F-actin of the HEK293ar cells, the staining of F-actin was faint and intense at the fringe of cells and the buds at their surface. The cytoskeletal proteins staining including actin、α-tubulin and plectin seen in resistant cells and their periphery was diffuse, and the staining at cell-cell contacts was intense in HEK293ar cell aggregates, whereas the staining in HEK293 cells were faint and diffuse in the cytoplasm. Plectin expression was shown to be elevated in HEK293ar cell spheroids compared with corresponding parental cells and plectin was clearly localized to cell-cell junctions and diffused to cytoplasm in HEK293ar cell spheroids, but this was much less obvious in parental cells. In addition, we showed that CD147 partially colocalized with plectin in anoikis-resistant cell clusters and this was especially evident at cell-cell contacts and cell edges (0 Conclusion:
1、An anoikis-resistant cell subpopulation named HEK293ar was successfully acquired ,which may be used as a cell model to study cell-cell contacts and cell spheroids growth of engineering cell.
2、A dual phase transient expression for protein production in HEK293ar cells was established successfully: transfection under adhesion condition and protein production in suspension. The anoikis-resistant HEK293ar may be suitable for transient gene expression production of protein and carrier-free immobilization culture.
3、The study reveals a novel role of CD147 in cell-cell adhesion during the process of anoikis resistance by using a natural anoikis-resistance model. Our results reinforce the observation that E-cadherin-PI3-K/Akt-dependent survival signals are activated by the CD147-mediated cell-cell contacts cell signaling.
4、CD147 may promote the organization of the aberrant cytoskeleton in anoikis-resistant HEK293ar cells through the plectin,a cytoskeleton network integrator.
通过建立导致HEK293细胞脱离培养时完全“失巢”的培养模型,首先通过包括电镜观察、TUNEL染色、流式检测、Hoechst33342-PI双染色等多种方法研究HEK293细胞在失巢培养条件下发生的凋亡现象;并经过悬浮和贴壁培养的多轮循环,获得一种具有悬浮适应-可贴壁特性、并且能够抵御失巢凋亡的HEK293ar亚群,进而通过多种方法对该细胞亚群的失巢凋亡抗性特性进行研究,并研究该亚群是否是通过建立细胞间连接形成细胞团进而赋予细胞失巢凋亡的抗性。其次,基于失巢凋亡抗性亚群具有的失巢凋亡抗性而以细胞团形式生存和可贴壁培养的双重特点,对其可能在建立新型瞬时转染系统生产蛋白的可行性和无载体固定化细胞团培养的增殖特性进行研究。再次,以在失巢条件下可形成细胞团而生存的失巢凋亡抗性亚群HEK293ar和失巢敏感的亲本细胞HEK293这一对自然细胞模型研究HAb18G/CD147黏附分子在失巢凋亡抗性细胞中的表达差异,并研究其调节细胞间连接的作用,进而明确HAb18G/CD147调节的细胞间连接在失巢凋亡抗性调节中的作用;同时还研究另外一种黏附分子E-cadherin是否也参与细胞失巢凋亡抗性的调节,并研究这两个黏附分子在失巢凋亡抗性调节中的可能相互联系,进而研究HAb18G/CD147调节的细胞连接在抑制失巢凋亡信号调节通路中的作用和信号调节网络。最后,基于失巢条件下,细胞形态和骨架发生的显著变化,研究失巢敏感HEK293细胞和抗性亚群HEK293ar细胞在失巢条件下主要骨架蛋白的重排方式的差异,并研究HAb18G/CD147和骨架蛋白组织分子plectin之间可能的相互作用,进而研究HAb18G/CD147是否通过plectin而调节骨架重排的可能性。
方法:根据研究目的,实验内容共分为四个部分,实验操作方法简述如下。
1哺乳动物工程细胞可贴壁失巢凋亡抗性亚群(HEK293ar)的获得建立完全失巢培养模型的培养条件,通过电镜观察等研究该细胞在失巢条件下由于发生失巢凋亡而死亡的时间规律;通过有限稀释法获得HEK293的单细胞克隆株,分别研究其在失巢条件下的失巢凋亡敏感情况;研究细胞失巢模型培养和细胞贴壁培养长时间循环培养对单克隆细胞群失巢凋亡抗性的影响,进而通过细胞失巢培养和贴壁培养的多轮循环以获得具有失巢凋亡抗性的可贴壁细胞亚群;长时间失巢培养观察失巢凋亡抗性亚群的细胞形态变化,以及不同时间取样检测其发生的失巢凋亡,以验证其具有失巢凋亡抗性的特性是否稳定存在;直接将失巢培养的细胞群在贴壁条件下培养,观察细胞是否还具有重新贴壁培养的特性。最终以获得一种具有以建立细胞间连接而成团具有失巢凋亡抗性的可贴壁性细胞亚群。
2、抗性细胞亚群HEK293ar细胞在瞬时转染法生产蛋白和无载体固定化方面的应用研究
通过Lipofectin2000试剂分别在失巢敏感细胞HEK293和抗性细胞HEK293ar中高效转染pEGFP/N1,悬浮培养研究其在悬浮培养条件下生产蛋白的差异;通过Lipofectin2000试剂在抗性细胞HEK293ar中转染pEGFP/N1,连续悬浮培养并每天取样研究其在悬浮条件下生产蛋白效率的变化。重要的,通过建立高效聚乙烯亚胺转(PEI)染技术,建立基于HEK293ar细胞亚群的高效PEI瞬时转染技术策略。
3、HAb18G/CD147黏附分子参与调节细胞连接和失巢凋亡抗性的研究
以获得的失巢凋亡抗性亚群HEK293ar和亲本细胞HEK293这对细胞为模型,通过流式细胞术测定和western blot等方法研究HAb18G/CD147黏附分子的表达差异,并研究其在悬浮培养抗性细胞时形成的细胞团中的亚细胞定位;通过RNAi技术下调HAb18G/CD147表达研究其对细胞间连接和细胞凋亡抗性的影响;研究参与调节细胞间连接和凋亡抗性的另外一种黏附分子E-cadherin在这对细胞间的表达差异,并通过RNAi技术研究下调此蛋白表达对细胞间连接和细胞存活的影响;通过western blot和免疫荧光法研究下调HAb18G/CD147对E-cadherin表达和细胞生存的影响;通过额外添加EDTA和抗-E-cadherin抗体阻断细胞间连接研究封闭或破坏E-cadherin作用对细胞表达HAb18G/CD147的影响,并通过RNAi技术下调E-cadherin表达研究其对HAb18G/CD147表达的影响。通过研究添加LY294002(PI-3K通路信号竞争性抑制剂)和PD98059(ERK通路抑制剂)等信号通路抑制剂对失巢凋亡抗性亚群细胞增殖和细胞间连接的影响,研究其参与失巢凋亡抗性调节的细胞主要信号通路。
4、失巢凋亡抗性亚群在失巢培养下的细胞骨架特征和黏附分子HAb18G/CD147之间的关系研究
通过形态观察研究抗性细胞亚群HEK293ar细胞在失巢条件的细胞连接和细胞成团现象;研究抗性细胞在失巢前后的微纤维丝细胞骨架的特征;通过免疫荧光法研究HEK293ar细胞在失巢培养下actin (G-或F-)、α-tubulin和plectin的骨架重排特征以及HAb18G/CD147的亚细胞定位;采用RNAi干涉
HAb18G/CD147表达研究其对F-actin骨架重排的可能影响;通过免疫荧光法研究失巢凋亡时HAb18G/CD147和plectin参与失巢凋亡调节的现象;通过免疫荧光双标记法研究HAb18G/CD147和plectin在失巢培养抗性亚群时的相互作用,通过RNAi下调HAb18G/CD147研究其对失巢培养时这两个分子相互作用的可能影响。
结果:
1、成功建立了针对失巢培养HEK293细胞的“完全失巢”模型,并成功用于研究工程细胞HEK293在悬浮条件下发生的失巢凋亡。观察到HEK293细胞在失巢条件下发生染色体断裂、膜发泡、DNA断裂、凋亡小体形成和亚二倍体峰等典型的细胞凋亡现象。得到八株HEK293细胞单克隆细胞,研究得知,亲本细胞是由一群有不同失巢敏感程度的细胞亚群而组成的。长时间失巢培养有助于提高细胞的抗失巢凋亡能力。通过利用细胞失巢培养和贴壁培养循环获得了一种具有失巢凋亡抗性以细胞团方式生长的可贴壁细胞亚群。该亚群具有稳定的失巢凋亡抗性和重新贴壁培养的的特性。
2、贴壁转染EGFP基因后,抗性细胞HEK293ar在悬浮条件下培养到第三天时仍然维持着高效生产蛋白的能力,而亲本细胞HEK293则由于发生失巢凋亡蛋白产率和HEK293ar相比有明显下降。长时间悬浮培养HEK293ar时,可以维持初始时的高效生产蛋白的能力。利用HEK293ar细胞进行无载体固定化培养时,维持细胞活性在80-87 %,比生长速率达到0.05-0.06 hr-1,此细胞亚群可以用于无载体固定化培养。建立了基于HEK293ar细胞亚群的高效PEI转染策略,其转染效率高达65-75 %,而且在悬浮状态时可以维持高效生产蛋白的能力。
3、利用失巢凋亡抗性细胞HEK293ar和HEK293细胞这对自然细胞研究模型,证实黏附分子HAb18G/CD147内源性表达水平的提高是抗性细胞具有失巢凋亡抗性的重要原因。下调HAb18G/CD147可以导致细胞失巢培养条件下细胞间连接断裂以及细胞发生失巢凋亡。成功利用此模型研究证实E-cadherin黏附分子也参与了抗性细胞的抗性调节。下调HAb18G/CD147可以导致E-cadherin在24 hr后逐步降解,细胞间连接断裂。利用EDTA溶液以及抗E-cadherin抗体阻断细胞间连接对HAb18G/CD147表达没有显著影响,干涉E-cadherin也对HAb18G/CD147表达没有明显影响。用PI-3K通路信号竞争性抑制剂LY294002处理抗性细胞对抗性细胞的增殖和细胞间连接有影响,而用ERK通路抑制剂PD98059处理却没有同样的效果。
4、阐明了HEK293ar在失巢培养条件下细胞间连接加强和微纤维丝骨架重排的现象;抗性细胞亚群HEK293ar骨架重排在失巢条件下和贴壁下相比呈现紊乱的F-actin排布形式( aberrant cytoskeleton distribution );通过免疫荧光法研究得知,HEK293ar细胞在失巢下细胞骨架蛋白actin、α-tubulin和plectin均呈现紊乱的重排形式,而且和亲本细胞相比,其表达均有所上调。下调HAb18G/CD147导致抗性细胞HEK293ar失巢条件下的F-actin发生重排,表达变弱,呈现典型的细胞发生凋亡时的骨架重排现象,在细胞边缘以及表面突起处部分表达变强;发现抗性细胞亚群在失巢培养时,plectin和HAb18G/CD147的表达存在部分共定位,两分子在抗性细胞中的表达呈正相关(0
1、成功获得具有失巢凋亡抗性的贴壁性HEK293ar细胞亚群,以用做研究细胞间连接和工程细胞团生长时的细胞模型,此细胞亚群的获得为建立高效便宜的瞬时转染系统奠定了细胞基础,也是进行无载体固定化培养的适宜细胞亚群。
2、成功建立了基于抗性细胞亚群HEK293ar的双阶段瞬时高效转染生产药物蛋白策略,证明了此细胞亚群适宜于用于建立高效转染细胞生产蛋白平台,并且也适宜作为一种重要的无载体固定化培养的细胞亚群。
3、利用此模型细胞研究了HAb18G/CD147黏附分子调节的细胞间连接抑制失巢凋亡抗性的信号通路,得出结论为:HAb18G/CD147调节的细胞连接是以E-cadherin-PI-3K依赖的信号通路调节失巢凋亡抑制的。
4、利用此模型研究了失巢抗性细胞在失巢下的细胞骨架重排特征,证实了失巢凋亡抗性细胞在失巢下过表达骨架蛋白的特性;HAb18G/CD147可能是通过骨架蛋白的组织者plectin调节细胞骨架的重排。
Aim:
First, to establish a cell detachment model for studying anoikis of HEK293 cell. And then to acquire an anoikis-resistant subpopulation of HEK293 and study the anoikis resistance by many methods. Second, based on the acquired subpopulation, to study the advantage and feasibility of this subpopulation in transient gene expression production of protein; and then to study the feasibility and proliferation of this subpopulation using as a carrier-free immobilization culture model. Third,using the pair of HEK293 and the acquired anoikis-resistant subpopulation, to study expression change of HAb18G/CD147,an adherence molecule, in the acquired subpopulation, and then explore the function of HAb18G/CD147-mediated cell-cell contact in the anoikis-resistance regulation; And then to explore the other adherence molecule, E-caherin, in the cell-cell contact and anoikis-resistance regulation. And, to knock-down the two molecule expression using RNAi respectively and further investigate their possible correlation in the anoikis-resistance regulation. Then, with signal transduction inhibitors to treat the anoikis-resistant subpopulation and explore the possible signal pathway of anoikis resistance regulation. Lastly, based on the morphology and cytoskeleton change of cell upon suspension, to explore the cytoskeletal rearrangement of suspended cells; And then to study the possible correlation of HAb18G/CD147 and plectin in the anoikis regulation; And then to down-regulate HAb18G/CD147 expression with RNAi and study the this effect on the plectin rearrangement in the anoikis-resistant subpopulation.
Methods: All the studies can be divided into four parts according to experiments’aims. The protocols were briefly described as the following:
1、To acquire an adhesive and anoikis-resistant subpopulation To establish the“homeless”cell-detachment culture model and study the anoikis of HEK293 cell upon suspension using many methods including TUNEL assay, ultrastructure assay by TEM and SEM , flow cytometer and Hoechst33342-PI staining. And then to explore the change of cell number resulting from the anoikis upon suspension using the Hoechst33342-PI staining. And, to acquire several single cell clones from parental HEK293 cell using limited dilution cloning and then to study sensitivity of these subpopulation to cell detachment; Then to evaluate the effect of long-term cell detachment culture on the anoikis resistance of single cell clone subpopulations; To acquire the anoikis-resistant and adhesive subpopulation of HEK293 cell with sequential cycles of adherent and suspended culture. And then to study the maintaining of anoikis resistance and possibility of re-adhesive culture.
2、To study the application of anoikis-resistant cell subpopulation in transient gene expression production of protein and carrier-free immobilization culture
To transfect the pEGFP/N1 in anoikis-resistant or -sensitive HEK293 cell under adhesive couture condition and then study the efficiency of these two cell subpopulation to produce protein under suspension condition. And to transfect the pEGFP/N1 in anoikis-resistant subpopulation using Lipofectin2000 and explore the production efficiency of anoikis-resistant HEK293 cell upon suspension. Secondly, to establish the PEI transfection strategy. Thirdly, to cultivate the anoikis-resistant subpopulation of HEK293 cell in suspension and study the feasibility and proliferation of this subpopulation using as a carrier-free immobilization culture model.
3、To study whether HAb18G/CD147-mediated cell-cell contacts involve in the anoikis-regulation
Using the acquired anoikis-resistant and -sensitive HEK293 cell as cell model, to study the expression change of HAb18G/CD147 using western blot and flow cytometer between these two HEK293 subpopulation and investigate HAb18G/CD147 subcellular localization in the anoikis-resistant subpopulation using immunofluorescence staining. And then to study the effect of down-regulation of HAb18G/CD147 on the cell-cell contacts and cell survival. To study the down-regulation of HAb18G/CD147 on the E-cadherin expression using immunofluorescence staining and western blot; Then to explore the effect of inhibition of cell-cell inhibition by EDTA-treatment and an additional blockade of E-cadherin binding by an anti-E-cadherin antibody inhibited the cell-cell contacts formation on the expression of HAb18G/CD147 and cell-cell contact formation; To knock-down the E-cadherin expression by RNAi and further explore the effect of down-regulation of E-cadherin on the HAb18G/CD147 expression and cell survival. Lastly, to treat cell culture with signal transduction inhibitors and then study whether the anoikis suppression of HAb18G/CD147–mediated cell-cell contacts might be mediated through either the Ras-ERK1/2 or PI3-K-Akt cascade.
4、To study the cytoskeleton rearrangement of anoikis-resistant subpopulation upon suspension and the correlation of HAb18G/CD147 and plectin
To study the reinforcement of cell-cell contacts and cell aggregates of anoikis resistant subpopulation using morphology detection; and then to explore the F-actin distribution in anoikis-resistant cell upon suspension and adhesion culture. To study the cytoskeleton rearrangement including actin (G-or F-)、α-tubulin and plectin and HAb18G/CD147 expression of anoikis-resistant subpopulation upon suspension using a laser scanning confocal microscope; And to explore the co-localization of plectin and HAb18G/CD147 expression using double immunofluorescence staining, and to down-regulate the HAb18G/CD147 expression and evaluate this effect on the co-localization or interaction of plectin and HAb18G/CD147 expression in cytoskeleton rearrangement during anoikis suppression.
Results:
1、A cell detachment culture model adapting to HEK293 cell was successfully established. Thus, the anoikis of HEK293 cell upon suspension was studied using several methods. In detached HEK293 cell, anoikis was confirmed using TUNEL assay and ultrastructure analysis of SEM and TEM; Fragmented DNA, cell shrinkage and dead cell was confirmed to be shown in suspended HEK293 cell; Additionally, HEK293 cell was confirmed to undergo anoikis upon suspension in a time-dependent manner. Using limited dilution cloning, eight single cell clones from parental HEK293 cell were acquired successfully and were shown to be sensitive to cell detachment differently. And then the long-term cell-detachment culture was shown to have positive effect on the anoikis-resistance of cell subpopulation. Lastly, an anoikis-resistant subpopulation of HEK293 cell ( named HEK293ar ) was acquired successfully with sequential cycles of adherent and suspended culture.HEK293ar was shown to form cell-cell contacts and resist to anoikis with respect to HEK293.The acquired HEK293ar cell was shown to form cell spheroids and grow again under adhesion condition.
2、The HEK293ar cell was shown to maintain high efficiency of protein production under suspension with respect to the parental cell. In addition, even 3 days post-trasfection,the cells still maintained high production potency. Importantly,based on the HEK293ar cells, a high-efficiency PEI-transfection strategy was established; The transfection efficiency was up to 65-75 % and a dual phase transient expression for protein production in HEK293ar cells was established successfully: transfection under adhesion condition and protein production in suspension. The HEK293ar cell was shown to grow as cell spheroids and the cell viability as cell spheroids in suspension culture had little change during carrier-free immobilization culture. The specific growth rate was 0.05-0.06 hr-1.
3、The endogenetic HAb18G/CD147 expression ,an adhesive molecule, was shown to be elevated in suspended HEK293ar compared to the parental cell. And HAb18G/CD147 expression was shown to be localized in the cell-cell contacts of HEK293ar cell using immunofluorescence staining; the down-regulation of HAb18G/CD147 expression resulted in the disruption of cell-cell contacts and cell death of HEK293ar cell in suspension. In addition, E-cadherin, a Ca-dependent adhesive molecule, was also shown to be elevated in suspended HEK293ar cell, and the knock-down of E-cadherin also resulted in the inhibition of cell-cell contacts and cell death of HEK293ar cell under suspension. The down-regulation of HAb18G/CD147 was shown to result in the degradation of E-cadherin and disruption of cell-cell contacts. However, inhibition of cell-cell contacts and down-regulation of E-cadherin had no such effect on the HAb18G/CD147 expression. LY294002μmol/L decreased cellular DNA content in cell spheroids in a dose-dependent manner assayed by the cell proliferation analysis kit (Newman-Keuls Multiple Comparison Test, 50 vs. 20, P < 0.001), whereas up to 50μmol/L ERK inhibitor, PD98059 had virtually no effect. A dose-response curve of LY294002 treatment versus cell proliferation confirmed the LY294002 effect over a wide concentration range in these spheroids. Moreover, cell-cell contact formation was dramatically inhibited by LY294002 but not PD98059.
4、The reinforcement of cell adhesion and cytoskeleton rearrangement were observed in suspended HEK293ar cell. The adherent cells showed elaborated microfilaments, and appeared flattened to the dish in light microscopy; however, the suspense cells showed a rounded morphology with a diffuse distribution of F-actin throughout the cytoplasm, appearing spherical and conglobate.The down-regulation of HAb18G/CD147 was shown to induce cell to undergo anoikis and F-actin rearrangement. The reorganization of plectin seemed to be consistent with features of apoptosis; weak staining of plectin concentrated at the site of apoptotic body’s formation might suggest importance of these proteins for this process. Additionally, apoptotic cells showed a different staining pattern with CD147 included in the apoptotic bodies during anoikis. Using F-actin microfilaments as a paradigm of cytoskeleton, we observed that the interference of CD147 caused changes in the organization of F-actin of the HEK293ar cells, the staining of F-actin was faint and intense at the fringe of cells and the buds at their surface. The cytoskeletal proteins staining including actin、α-tubulin and plectin seen in resistant cells and their periphery was diffuse, and the staining at cell-cell contacts was intense in HEK293ar cell aggregates, whereas the staining in HEK293 cells were faint and diffuse in the cytoplasm. Plectin expression was shown to be elevated in HEK293ar cell spheroids compared with corresponding parental cells and plectin was clearly localized to cell-cell junctions and diffused to cytoplasm in HEK293ar cell spheroids, but this was much less obvious in parental cells. In addition, we showed that CD147 partially colocalized with plectin in anoikis-resistant cell clusters and this was especially evident at cell-cell contacts and cell edges (0
1、An anoikis-resistant cell subpopulation named HEK293ar was successfully acquired ,which may be used as a cell model to study cell-cell contacts and cell spheroids growth of engineering cell.
2、A dual phase transient expression for protein production in HEK293ar cells was established successfully: transfection under adhesion condition and protein production in suspension. The anoikis-resistant HEK293ar may be suitable for transient gene expression production of protein and carrier-free immobilization culture.
3、The study reveals a novel role of CD147 in cell-cell adhesion during the process of anoikis resistance by using a natural anoikis-resistance model. Our results reinforce the observation that E-cadherin-PI3-K/Akt-dependent survival signals are activated by the CD147-mediated cell-cell contacts cell signaling.
4、CD147 may promote the organization of the aberrant cytoskeleton in anoikis-resistant HEK293ar cells through the plectin,a cytoskeleton network integrator.
引文
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