The Chinese hamster ovary (CHO) cell is the most representative mammalian cell protein expression system, and it is widely used in recombinant protein, vaccine and other biopharmaceutical fields. However, due to its vulnerability to environmental factors, apoptosis, and metabolic inhibitors, CHO cells demonstrate poor robustness, and thus the integrated viable cell density and unit cell productivity are largely limited. To improve the robustness and foreign protein expression efficiency of CHO cells, we employed CRISPR/Cas9 to knock out the apoptosis genes Bax and Bak and the lactate dehydrogenase gene LDHa, thereby blocking apoptosis and lactic acid metabolism pathways. The results of apoptosis and single cell viability detection showed that the number of apoptotic cells in the knockout cell lines Bax^-/-, Bax-bak^-/-, and LDHa-Bax-bak^-/- was reduced by 22.51%, 37.73%, and 64.12%, respectively, compared with the wild-type cell line CHO-K1, which indicated that the anti-apoptotic ability was significantly improved. After staurosporine treatment, the single cell viability of Bax^-/-, Bax-bak^-/-, and LDHa-Bax-bak^-/- cells was increased by 30.8%, 22%, and 41.1%, respectively. After treatment with puromycin, the single cell viability of Bax^-/-, Bax-bak^-/-, and LDHa-Bax-bak^-/- cells was increased by 26.7%, 30.7%, and 38.8%, respectively. To further investigate the production performance of cells obtained after blocking apoptosis and lactic acid metabolism pathways, we induced transient expression of human tissue plasminogen activator (tPA) in these cells. The results showed that the secretion of tPA in Bax^-/-, Bax-Bak^-/-, and LDHa-Bax-Bak^-/- cells was 11.12%, 46.18%, and 63.13%, respectively, higher than that in wild-type CHO-K1 cells. The expression of intracellular tPA was increased by 35.65%, 130%, and 192.15%. In conclusion, blocking apoptosis and lactic acid metabolism pathways simultaneously can improve cell robustness and productivity, with the performance better than blocking the apoptosis pathway alone. The above results indicated that the constructed cell lines were expected to be the delivery carriers of protein drugs such as medicinal peptides, and better used for the treatment of diseases.
CHO Cells ; Cricetulus ; Animals ; Apoptosis/genetics* ; Lactic Acid/metabolism* ; Recombinant Proteins/biosynthesis* ; L-Lactate Dehydrogenase/genetics* ; bcl-2-Associated X Protein/genetics* ; bcl-2 Homologous Antagonist-Killer Protein/genetics* ; Cricetinae ; CRISPR-Cas Systems ; Staurosporine/pharmacology*

Objective To explore the mechanobiological mechanism of fluid shear force(FSF)on the protection,injury,and destruction of the structure and function of the blood-brain barrier(BBB)under normal physiological conditions,ischemic hypoperfusion,and postoperative hyperperfusion conditions.BBB is mainly composed of brain microvascular endothelial cells.Rat brain microvascular endothelial cells(rBMECs)were used as model cells to conduct the investigation.Methods rBMECs were seeded at a density of 1×105 cells/cm2 and incubated for 48 h.FSF was applied to the rBMECs at 0.5,2,and 20 dyn/cm2,respectively,simulating the stress BBB incurs under low perfusion,normal physiological conditions,and high FSF after bypass grafting when there is cerebral vascular stenosis.In addition,a rBMECs static culture group was set up as the control(no force was applied).Light microscope,scanning electron microscope(SEM),and laser confocal microscope(LSCM)were used to observe the changes in cell morphology and cytoskeleton.Transmission electron microscope(TEM)was used to observe the tight junctions.Immunofluorescence assay was performed to determine changes in the distribution of tight junction-associated proteins claudin-5,occludin,and ZO-1 and adherens junction-associated proteins VE-cadherin and PECAM-1.Western blot was performed to determine the expression levels of tight junction-associated proteins claudin-5,ZO-1,and JAM4,adherens junction-associated protein VE-cadherin,and key proteins in Rho GTPases signaling(Rac1,Cdc42,and RhoA)under FSF at different intensities.Results Microscopic observation showed that the cytoskeleton exhibited disorderly arrangement and irregular orientation under static culture and low shear force(0.5 dyn/cm2).Under normal physiological shear force(2 dyn/cm2),the cytoskeleton was rearranged in the orientation of the FSF and an effective tight junction structure was observed between cells.Under high shear force(20 dyn/cm2),the intercellular space was enlarged and no effective tight junction structure was observed.Immunofluorescence results showed that,under low shear force,the gap between the cells decreased,but there was also decreased distribution of tight junction-associated proteins and adherens junction-associated proteins at the intercellular junctions.Under normal physiological conditions,the cells were tightly connected and most of the tight junction-associated proteins were concentrated at the intercellular junctions.Under high shear force,the gap between the cells increased significantly and the tight junction and adherens junction structures were disrupted.According to the Western blot results,under low shear force,the expression levels of claudin-5,ZO-1,and VE-cadherin were significantly up-regulated compared with those of the control group(P<0.05).Under normal physiological shear force,claudin-5,ZO-1,JAM4,and VE-cadherin were highly expressed compared with those of the control group(P<0.05).Under high shear force,the expressions of claudin-5,ZO-1,JAM4,and VE-cadherin were significantly down-regulated compared with those of the normal physiological shear force group(P<0.05).Under normal physiological shear force,intercellular expressions of Rho GTPases proteins(Rac1,Cdc42,and RhoA)were up-regulated and were higher than those of the other experimental groups(P<0.05).The expressions of Rho GTPases under low and high shear forces were down-regulated compared with that of the normal physiological shear force group(P<0.05).Conclusion Under normal physiological conditions,FSF helps maintain the integrity of the BBB structure,while low or high shear force can damage or destroy the BBB structure.The regulation of BBB by FSF is closely related to the expression and distribution of tight junction-associated proteins and adherens junction-associated proteins.

Recent years, the incidence and mortality of prostate cancer have increased dramatically in China. At earlier stages, most diagnosed prostate cancers are responsive to androgen depletion treatment, yet, nearly all patients will eventually progress to metastatic androgen-independent prostate cancer (AIPC), which still has no effective therapeutic method or drug to deal with. 11'-Deoxyverticillin A (C42) belongs to the family of epipolythiodioxopiperazines (ETPs), an interesting class of fungal toxins that inhibit farnesyl transferase. Compounds holding such a property have been explored as putative anticancer agents. In this study, using PC3M cells, an AIPC cell line, we investigated the effect of the compound on apoptosis and explored the underlying mechanism. It revealed that C42 markedly enhanced the activity of caspase-3/7 and increased the accumulation of the cleaved PARP, all of which are the markers of apoptosis. It also revealed that C42 either decreased cell viability or inhibited the growth of PC3M cells. Moreover, we observed that the loss of cell viability and cell growth inhibition induced by C42 were both time- and dosage dependent. Taken together, we indicated that C42 can induce caspase-dependent apoptosis in AIPC cells, and the results presented here will broaden our knowledge about the molecular mechanisms by which C42 exerts its anticancer activity, and future work in this direction may provide valuable information in the development of these compounds into effective cancer therapeutic strategies against androgen-independent prostate cancer.
Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Caspase 7 ; metabolism ; Cell Line, Tumor ; Disulfides ; pharmacology ; Farnesyltranstransferase ; antagonists & inhibitors ; Humans ; Male ; Mycotoxins ; pharmacology ; Piperazines ; pharmacology ; Prostatic Neoplasms ; pathology

Objective To observe the effect of Glutsmine on intestinal barrier function after traumatic brain injury in rats. Methods A total of 54 adult male Wistar rats were divided randomly into 3 groups, ie, normal group (Group N, 6 rats), TBI group (Group T, 24 rats) and Giutamine intervention group (Group G, 24 rats). Group T and Group G were subdivided into 4 groups according to detection time at days 1,3, 5 and 7 respectively. Meanwhile, 6 rats were enlisted in each group. The intestinal mucosa structure was detected by histopathological examination and electron microscopy. Apoptosis was detected by in situ immunohistochemical staining (TUNEL). Results Glutamine could relieve the pathological lesion of gut mucosa and decrease intestinal mucosa cell apoptosis after traumatic brain injury. Conclusion Glutamine can protect intestinal mucosa barrier function following traumatic brain injury.