The Suspension-adapted-adherent Cell Subpopulation (HEK293ar) Acquisition And Survival Mechanism Study Of Mammalian Engineering Cell

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 cultureTo 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-regulationUsing 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 plectinTo 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