Anticancer property of sediment actinomycetes against MCF-7 and MDA-MB-231 cell lines

ObjectiveTo investigate the anticancer property of marine sediment actinomycetes against two different breast cancer cell lines.MethodsIn vitro anticancer activity was carried out against breast (MCF-7 and MDA-MB-231) cancer cell lines. Partial sequences of the 16s rRNA gene, phylogenetic tree construction, multiple sequence analysis and secondary structure analysis were also carried out with the actinomycetes isolates.ResultsOf the selected five actinomycete isolates, ACT01 and ACT02 showed the IC50 value with (10.13±0.92) and (22.34±5.82) μg/mL concentrations, respectively for MCF-7 cell line at 48 h, but ACT01 showed the minimum (18.54±2.49 μg/mL) level of IC50 value with MDA-MB-231 cell line. Further, the 16s rRNA partial sequences of ACT01, ACT02, ACT03, ACT04 and ACT05 isolates were also deposited in NCBI data bank with the accession numbers of GQ478246, GQ478247, GQ478248, GQ478249 and GQ478250, respectively. The phylogenetic tree analysis showed that, the isolates of ACT02 and ACT03 were represented in group I and III, respectively, but ACT01 and ACT02 were represented in group II. The multiple sequence alignment of the actinomycete isolates showed that, the maximum identical conserved regions were identified with the nucleotide regions of 125 to 221st base pairs, 65 to 119th base pairs and 55, 48 and 31st base pairs. Secondary structure prediction of the 16s rRNA showed that, the maximum free energy was consumed with ACT03 isolate (-45.4 kkal/mol) and the minimum free energy was consumed with ACT04 isolate (?7.6 kkal/mol).ConclusionsThe actinomycete isolates of ACT01 and ACT02 (GQ478246 and GQ478247) which are isolated from sediment sample can be further used as anticancer agents against breast cancer cell lines.

REVERSION OF MULTIDRUG RESISTANCE IN THE P-GLYCOPROTEIN POSITIVE BREAST CANCER CELL LINE(MCF-7/ADR) BY INTRODUCTION OF HAMMERHEAD RIBOZYME

A hammerhead ribozyme which site-specifically cleaved the GUC position in canon 880 of the mdr1 mRNA was designed. The target site was chosen between the two ATP binding sites, which may be important for the function of the P-Gp as an ATP-dependent pump. A DNA sequence encoding the ribozyme gene was then incorporated into a eukaryotic expression vector (pH Apr-1 neo) and transfected into the breast cancer cell line MCF-7/Adr, which is resistant to adriamycin and expresses the MDR phenotype. The ribozyme was stably expressed in the cell line by the RNA dot blotting assay. The result of Northern blot assay showed that the expressed ribozyme could decrease the level of mdrl mRNA expression by 83. 5 %; and the expressed ribozyme could inhibite the formation of p-glycoprotein detected by immuno- cy-tochemistry assay and could reduce the cell’s resistance to adrimycin; this means that the resistant cells were 1 000-fold more resistant than the parental cell line(MCF-7), whereas those cell clones that showed ribozyme expression were only 6-fold more resistant than the parental cell line. These results show that a potentially useful tool is at hand which may inactivate MDR1 mRNA and revert the multidrug resistance phenotype.

Studies on mechanism of cis9,trans11-CLA and trans10,cis12-CLA inducing apoptosis of human breast cancer cell line MCF-7

Objective： The aim of the study was to explore the activities of cis9, trans11-CLA （C9, t11-CLA） and transl0, cis12-CLA （t10, c12-CLA） inhibiting tumor, and investigate their relationships with PPARy and apoptotic proteins, and mechanism of anti-cancer. Methods： The inhibitory rate, cell growth curve and apoptotic morphological observation of MCF-7 cells were obtained by MTT assay, trypan blue staining and Hoechst33342 fluorescence staining. The apoptotic rate and cell cycle were detected with flow cytometry. Transcriptional level of genes was detected with RT-PCR semi-quantitative method, and Western blot was performed to detect proteins levels. Results： The two CLA isomers could reduce cell proliferation （P 〈 0.05）, increase apoptotic rate （P 〈 0.05）, and increase obviously the transcriptional and protein levels of PPARy （P 〈 0.01）. The synchronism and correlation between the effects of CLA to PPARy and apoptotic proteins Bax, Bcl-2, Caspase 3 changes were found with the dose- and time-dependent manners. There was cooperative relation between the levels of PPARy and the rates of Bax/Bcl-2, Caspase 3 （small fragment） by experiments of PPARy inhibitor GW9662 and ligand Rosiglitazone. Conclusion： The apoptotic pathway of PPARy-Bcl-2-Caspase 3 signaling was found. The C9, t11-CLA and tl0, c12-CLA could inhibit MCF-7 cell proliferation and promote apoptosis via activating PPARy-Bcl-2-Caspase 3 pathway. CLA may be a kind of activator of PPARv.

Construction of Cox7a2 fluorescent vector and its effect on cytochrome C oxidase activity in mouse Sertoli cell line TM4

Objective To construct Cox7a2 fluorescent vector and study its effect on cytochrome C oxidase ( COX) activity in mouse Sertoli cell line TM4. Methods The coding region of CoxTa2 was amplified from mouse Sertoli cell line TM4 by RT-PCR. PCR product was

Anticancer activity of Tephrosia purpurea and Ficus religiosa using MCF 7 cell lines

Objective:To investigate anticancer activity of different fractions of Tephrosia purpurea[TP] （Sharapunkha,Fabaceae） and Ficus religiosa[FR]（Peepal,Moraceae）.Methods:The fractions of TP and FR were prepared and tested for in vitro anticancer activity using human MCF 7 cell line by trypan blue exclusion method.Results:The result showed that among all these fractions of TPI.TPIII.FRI and FRIII showed better anticancer activity compared to other fractions.The IC50 value for TPI（152.4μM）,TPIII（158.71μM）.FRI（160.3μM） and for FRIII（222.7μM） was observed.Conclusions:The present study shows anticancer potential of TP and FR fractions in MCF 7 cell line.

Sensitivity Evaluation of Two Human Breast Cancer Cell Lines to Tamoxifen through Apoptosis Induction

Tamoxifen citrate (TAM) has been used to treat breast cancer in women for many years. The com-parative effects of TAM in inducing apoptosis were evaluated in estrogen receptor-positive (ER- positive MCF-7) and estrogen receptor-negative (ER-negative MDA-MB-231) human breast cancer cell lines in vitro in order to determine if these two cell lines differ in their sensitivity to TAM. Mi-tochondrial membrane permeability potential disruption was assessed in both cell lines by a lip-ophilic cationic dye (DePsipher assay, Trevigen, Inc.) utilizing fluorescence microscopy. Using this specific fluorochrome, we were able to associate mitochondrial membrane disruption to early, mid-, and late apoptotic cells. TAM induced cell death via apoptosis in both ER-positive and ER- negative cells, however, apoptosis induction was more pronounced in ER-positive MCF-7 compared to ER-negative MDA-MB-231 breast cancer cells. These findings may have some therapeutic use in the treatment of estrogen dependent and estrogen independent breast cancer.

Effects of 4-(3-Chloro-Benzyl)-6,7-Dimethoxy-Quinazoline on Kinetics of P120-Catenin and Periplakin in Human Buccal Mucosa Squamous Carcinoma Cell Line

In order to detect molecular markers for the epidermal growth factor inhibitor 4-(3-chloro-benzyl)- 6,7-dimethoxy-quinazoline (tyrphostin), we investigated the kinetics of p120-catenin and periplakin in the human buccal mucosa squamous cancer cell line BICR 10 treated with 3 nM tyrphostin. Growth of BICR 10 cells was inhibited by treatment with tyrphostin. Although changes were not observed in the expression of EGFR and p120-catenin, expression of Akt, Src and periplakin in BICR 10 treated with 3 nM tyrphostin tended to decrease. In addition, phosphorylation of EGFR, Akt and Src was inhibited by treatment with tyrphostin. On immunocytochemical staining, immunoreactions with phosphorylated EGFR, phosphorylated Akt and phosphorylated p120-catenin were weak in BICR 10 treated with tyrphostin. There was a slight immunocy to chemical reaction to periplakin in BICR 10 cells induced by tyrphostin. In conclusion, the decrease in phosphorylation in EGFR and p120-catenin by tyrphostin, following the decrease in Src or Akt phosphorylation, may inhibit expression of several growth factors associated with the proliferation and migration of cancer cells.

Effect of 1,25(OH)_2D_3 on the growth and apoptosis of breast cancer cell line MCF-7

Objective To study the effect of 1,25 dihydroxyvitamin D 3 (1,25(OH) 2D 3) on the growth and apoptosis of breast cancer cell line MCF 7 Methods Cell number was determined using the MTT method Flow cytometric analysis was performed on cell cycles, and the percentage of apoptosis was counted Apoptotic cells were quantified by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL), and bcl 2 protein expression was estimated with Western blotting Results After incubation with 1,25(OH) 2D 3 10 7 mol/L for 48 hours, MCF 7 cells exhibited significant growth in a dose and time dependent manner Flow cytometric analysis indicated that cell numbers in G 0/G 1 increased along with increasing apoptotic peak and percentage With microscope and electron microscope observation, characteristics of apoptosis such as typical apoptotic bodies were commonly found TUNEL also showed that 1,25(OH) 2D 3 10 8 mol/L and 10 7 mol/L groups had significantly high apoptosis percentage than control group with dose dependence on induction apoptosis And Western blot showed that 1,25(OH) 2D 3 10 8 mol/L could down regulate bcl 2 protein and 10 7 mol/L could almost block bcl 2 protein expression Conclusions 1,25(OH) 2D 3 can inhibit cell growth with G 0/G 1 arrest, enhance the proliferation inhibition action of adriamycin, and induce apoptosis which may result from the down regulation of the anti apoptotic bcl 2 protein

STUDY OF ENHANCED IMMUNOGENECITY OF B7-1 GENE TRANSFECTED HUMAN HELA CELL LINE

This work was supposed by CMB (No. 96—635) This is one of papers of the special issue on gene therapy research (Chin J Cancer Res Vol. 9 No. 4 December, 1997). Although cervical carcinoma cells may express the human papillomavirus protein E6 and E7, they fail to induce an effective specific cytotoxic T lymphocyte response. Recent studies suggest that expression of CD 80 (B7 1) on tumor cells is effective to induce antitumor immune responses. 1,2 In our study, CD 80 gene was transfected into human Hela cell line with a CD 80 expression plasmid (B7 1 +pcDNA 3) by electroporation, then the immunogenecity of the modified Hela cell was tested in TLMC (tumor lymphocyte mixed culture) system. Thymidine lymphocyte proliferation assays showed that the response of human peripheral blood lymphocytes (PBLS) to CD 80 positive Hela cells demonstrated a substantial increase in cell proliferation compared to the response to control cells. Cocultivation of allogeneic PBLs with CD 80 positive tumor cells for three days can induce an increased secretion of IL 2. Our results demonstrate an immunostimulatory effect of CD 80 expression on cervical cancer cells, which provides a basis for the development of a therapeutic tumor vaccine.

Study on regulating mechanisms of oxocrebanine obtained from Stephania hainanensis H.S.Lo et Y.Tsoong on microtubule sites and tubulin in human breast cancer MCF-7 cells

Objective:To determine the destructive ability of oxocrebanine,an anti-breast cancer active compound obtained from Stephania hainanensis H.S.Lo et Y.Tsoong,on microtubule network,and investigate the effect of oxocrebanine on microtubule network homeostasis at both molecular and cellular levels.Methods:the EBI site competition method and molecular docking method were used to determine the occupation of the microtubule site of oxocrebanine.Western Blot was used to detect the effect of oxocrebanine on microtubule-associated proteins including STAT3,PAK1,CAMK4,and PKA.Results:The results of EBI site competition assay showed that the binding of EBI toβ-Tubulin covalent fusions produced adducts that appeared in regions of lower molecular weight thanβ-tubulin(ctrl 2).Molecular docking results showed that oxocrebanine could occupy the colchicine site of microtubule proteins.As revealed by Western Blot,the expression of STAT3 protein was decreased after MCF-7 cells have been treated with low,medium,and high concentration of oxocrebanine or the positive drug taxol for 48 h(P<0.01).The expression levels of PAK1 and Camk4 proteins aslo showed significant reductions(P<0.05,or P<0.01).Oxocrebanine also decreased the PKA protein in MCF-7 cells compared to the control group(P<0.01).Conclusions:Oxocrebanine,a ligand that binds at the colchicine site of tubulin,perturbs tubulin polymerization and causes mitosis in MCF-7 cells,thus leading to MCF-7 cell death.Oxocrebanine may promote microtubule dynamics through stathmin by inhibiting the expression levels of STAT3,PAK1,Camk4,and PKA proteins in MCF-7 cells.Oxocrebanine interfers with spindle formation,and ultimately causes mitotic catastrophe in MCF-7 cells.

Hesperidin as a preventive resistance agent in MCF-7 breast cancer cells line resistance to doxorubicin

Objective:To evaluate of hesperidin to overcome resistance of doxorubicin in MCF-7 resistant doxorubicin cells(MCF-7/Dox)in cytotoxicity apoptosis and P-glycoprotein(Pgp)expression in combination with doxorubicin.Methods:The cytotoxic properties.50%inhibition concentration(IC_(50))and its combination with doxorubicin in MCF-7 cell lines resistant to doxorubicin(MCF-7/Dox)cells were determined using MTT assay.Apoptosis induction was examined by double staining assay using ethidium bromide-acridine orange.Immunocytochemistry assay was performed to determine the level and localization of Pgp.Results:Single treatment of hesperidin showed cytotoxic activity on MCF-7/Dox cells with IC_(50)value of 11μmol/L.Thus,combination treatment from hesperidin and doxorubicin showed addictive and antagonist effect(CI>1.0).Hesperidin did not increase the apoptotic induction,but decreased the Pgp expressions level when combined with doxorubicin in low concentration.Conclusions:Hesperidin has cytotoxic effect on MCF-7/Dox cells with IC_(50)of 11μmol/L.Hesperidin did not increased the apoptotic induction combined with doxorubicin.Cochemotherapy application of doxorubicin and hesperidin on MCF-7/Dox cells showed synergism effect through inhibition of Pgp expression.

Tetrandrine:A Potent Abrogator of G_2 Checkpoint Function in Tumor Cells and Its Mechanism

Objective To assess the ability of tetrandrine （Tet） to enhance the sensitivity to irradiation and its mechanism in cell lines of human breast cancer p53-mutant MCF-7/ADR, p53-wild-type MCF-7 and human colon carcinoma p53-mutant HT-29 as well as in C26 colorectal carcinoma-bearing BALB/c mice. Methods MCF-7/ADR, HT-29 and MCF-7 cells were exposed to irradiation in the absence or presence of tetrandrine. The effect of Tet on the cytotoxicity of X-irradiation in these three cells was determined and the effect of tetrandrine on cell cycle arrest induced by irradiation in its absence or presence was studied by flow cytometry. Moreover, mitotic index measurement determined mitosis of cells to enter mitosis. Western blotting was employed to detect cyclin B1 and Cdc2 proteins in extracts from irradiated or non-irradiated cells of MCF-7/ADR, HT-29 and MCF-7 treated with tetrandrine at various concentrations. Tumor growth delay assay was conducted to determine the radio-sensitization of tetrandrine in vivo. Results Clonogenic assay showed that tetrandrine markedly enhanced the lethal effect of X-rays on p53-mutant MCF-7/ADR and HT-29 cells and the sensitization enhancement ratio （SER） of tetrandrine was 1.51 and 1.63, but its SER was only 1.1 in p53-wt MCF-7 cells. Irradiated p53-mutant MCF-7/ADR and HT-29 cells were only arrested in G2/M phase while MCF-7 cells were arrested in G1 and G2/M phases. Radiation-induced G2 phase arrests were abrogated by tetrandrine in a concentration-dependent manner in MCF-7/ADR and HT-29 cells, whereas redistribution within MCF-7 cell cycle changed slightly. The proportion of cells in M phase increased from 1.3% to 14.7% in MCF-7/ADR cells, and from 1.5% to 13.2% in HT-29 cells, but 2.4% to 7.1% in MCF-7 cells. Furthermore, the levels of cyclin B 1 and Cdc2 expression decreased after X-irradiation in MCF-7/ADR and HT-29 cells, and the mitotic index was also lower. Tet could reverse the decrease and induce the irradiated cells to enter mitosis （M phase）. Endosomatic experiment showed that tetrandrine caused tumor growth delay in irradiated mice. Conclusion Tetrandrine boosts the cell killing activity of irradiation both in vitro and in vivo. Tetrandrine is a potent abrogator for G2 checkpoint control and can sensitize the cells to radiation.

促分裂原活化蛋白激酶信号阻断剂在TPO促进UT7细胞增殖和分化中的作用

目的探索促分裂原活化蛋白激酶(mitogenactivatedproteinkinase,MAPK)信号阻断剂对巨核细胞生长分化因子(TPO)促进UT7细胞增殖和分化作用,阐明TPO对UT7细胞作用的机制。方法将EGFPpMSCV和MEK1pMSCV质粒转染UT7细胞;转染细胞与TPO共培养,Westernblot法检测UT7细胞MEK1(MAPK/Erkkinase1)磷酸化;观察转染突变(Ser222A)的MEK1和MAPK阻断剂PD98059对UT7细胞增殖的影响;采用流式细胞仪检测转染突变(Ser222A)的MEK1和MAPK阻断剂PD98059对UT7细胞表达CD41的影响。结果①重组逆转录病毒载体MEK1pMSCV和EGFPpMSCV转染UT7细胞,EGFP阳性率为60.73%。②TPO不同的作用时间(1h,3h),实验组磷酸化MEK1均低于对照组(P值均<0.05)。③MAPK阻断剂PD98059和外源突变MEK基因阻断了TPO对UT7细胞的增殖作用,DMSO对照组的细胞增殖率为98.58%,PD98059组的细胞增殖率为39.00%(P<0.05);转染EGFPpMSCV组细胞增殖率为102.12%,转染MEK1pMSCV组细胞增殖率为48.93%(P<0.05)。④MAPK阻断剂PD98059组UT7细胞CD41表达(10.27%)明显低于对照组(36.43%),转染MEK1pMSCV组UT7细胞CD41表达(24.03%)明显低于转染EGFPpMSCV组(40.16%)。结论TPO诱导UT7细胞MEK1磷酸化,TPO作用时间与UT7细胞MEK1的磷酸化有明显的关系。

SOX7靶向ERK1/2/PD-L1通路抑制结直肠癌血管生成

目的探讨性别决定区Y框蛋白7(SOX7)对结直肠癌血管生成的影响及潜在作用机制。方法应用免疫荧光检测结直肠癌患者组织样本中SOX7表达水平,之后通过裸鼠、转染SOX7 mimic的人结直肠癌细胞系SW480细胞和人脐静脉内皮细胞(HUVEC)共培养进一步研究。用Western-blot验证SOX7与ERK1/2/PD-L1对结直肠癌细胞的相关蛋白表达的影响。用CCK8检测SOX7与ERK1/2/PD-L1对HUVEC增殖的影响。通过体外内皮细胞成管实验测定SOX7与ERK1/2/PD-L1对肿瘤血管生成的影响。结果SOX7在人结直肠癌组织中表达被抑制(P<0.01),同时SOX7的过表达抑制了小鼠体内肿瘤生长(P<0.01)。SW480细胞中SOX7的过表达抑制了ERK1/2、c-Jun的表达,并在ERK1/2的激动剂Senkyunolide I的作用下上调了SW480细胞的ERK1/2、c-Jun蛋白表达(P<0.01),逆转了SOX7对SW480细胞中ERK1/2、c-Jun蛋白表达的影响(P<0.01)。HUVEC中SOX7抑制了PD-L1、V-EGFR2、p-PI3K、HIF-1α的蛋白表达,Senkyunolide I上调了HUVEC的PD-L1、V-EGFR2、p-PI3K、HIF-1α的蛋白表达,并逆转了SOX7对HUVEC中上述相关蛋白表达的影响(P<0.01)。PD-1/PD-L1 Inhibitor 3抑制了PD-L1、V-EGFR2、p-PI3K、HIF-1α的蛋白表达,SOX7过表达在PD-1/PD-L1 Inhibitor 3的影响下并没有表现出抑制作用。CCK8实验结果显示SOX7过表达显著抑制了HUVEC的增殖能力,Senkyunolide I作用下的两组HUVEC增殖能力较SOX7 NC组与SOX7 mimic组明显上升,PD-1/PD-L1 Inhibitor 3作用下的两组HUVEC增殖能力较SOX7 NC组与SOX7 mimic组明显下降,以上均有明显统计学差异(P<0.01)。成管实验结果显示SOX7过表达抑制了HUVEC的血管生成,Senkyunolide I强烈加速了血管生成,而PD-1/PD-L1 Inhibitor 3血管生成则被显著抑制,以上均有明显统计学差异(P<0.01)。结论SOX7通过ERK1/2/PD-L1通路抑制结直肠肿瘤的增殖和血管生成,SOX7可能是晚期CRC患者临床治疗中潜在的抗血管生成靶点。

Retinoic acid affects basic cellular processes and SOX2 and SOX18 expression in breast carcinoma cells

Genetic and molecular heterogeneity,together with intrinsic and acquired resistance to therapy,represent the major obstacles to the successful treatment of different types of breast carcinoma.Increasing evidence demonstrates that SOX transcription factors in breast carcinomas could act both as oncogenes and tumor suppressors and have been associated with tumor stage and grade,poor prognosis,and therapy resistance.Both SOX2 and SOX18 overexpression has been correlated with poor prognosis in breast carcinomas,and these genes are recognized as potential antitumor targets.Our aim was to evaluate the effect of retinoic acid(RA),a well-known cyto-differentiating agent,on breast carcinoma cells in vitro and to investigate the potential of RA treatment to modify the expression of SOX2 and SOX18 genes.By applying various experimental approaches,we evaluated the effect of RA on basic cellular processes in SK-BR-3 and MCF7 breast carcinoma cell lines.We have shown that RA inhibits cell growth,reduces the number of Ki-67 positive cells,and causes cell-cycle arrest.RA effect was more prominent in SK-BR-3 cell line that lacks SOX2 expression,including a higher decrease in cell viability,reduction in colony formation,and significant remodeling of cellular structure.We have shown that RA treatment led to the downregulation of SOX2 expression in MCF7 cells and to the reduction of SOX18 expression in both cell lines.By functional analysis,we showed that the anti-proliferative effect of RA in both cell lines was not based on the activity of stemness marker SOX2,pointing to a SOX2-independent mechanism of action.The ability of RA to reduce SOX2/SOX18 expression raises the possibility that these genes can be used as biomarkers to distinguish RA-responders from non-responders.Together,our study shows that the response of breast carcinoma cell lines to RA treatment may vary,highlighting that the development of RA-based therapy should consider differences in breast carcinoma subtypes.

Establishment of primary cultures of craniopharyngioma cells

Craniopharynigoma samples were collected from 36 patients. Out of the 36 samples, 29 achieved successful sub-culturing, with a success rate of 80.6%. Immunohistochemistry staining showed that cytokeratin-7 was positively expressed in the cytomembrane and cytoplasm of craniopharyngioma cells at 6-8 passages, confirming that all cultured cells were squamous epithelial cells. The doubling time of craniopharyngioma cells was 3 days, as confirmed by the 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide assay. In this study, craniopharyngioma cells cultured in vitro were established; however, establishment of immortalized craniopharyngioma cell lines requires further research.

应用C-to-G碱基编辑器对PCSK9基因靶向敲除的研究

目的:使用C-to-G碱基编辑器(CGBE)Td-CGBE-NG靶向敲除huh-7肝癌细胞系前蛋白转化酶枯草溶菌素9(PCSK9)基因,以CBE(AncBE4max)为参照,评价Td-CGBE-NG对该基因的敲除效果。方法:使用引入终止密码子的策略敲除huh-7肝癌细胞系PCSK9基因。构建碱基编辑器Td-CGBE-NG和AncBE4max,构建重组质粒sg386和sg555,将Td-CGBE-NG和sg386,AncBE4max和sg555分别共转染huh-7细胞,实现c.1447C>G和c.1953C>T的转换,在两位点引入终止密码子S386X(TGA)和Q555X(TAG),使用sanger测序和T载体克隆验证编辑效率;实时荧光定量聚合酶链反应(RT-qPCR)和蛋白免疫印迹法(Western blot)检测PCSK9基因mRNA和蛋白表达水平的变化。结果:sanger测序和T载体克隆结果显示,Td-CGBE-NG和AncBE4max的编辑效率分别为c.1447C>G 33%和c.1953C>T 25%;RT-qPCR结果显示,Td-CGBE-NG使PCSK9 mRNA的表达量降低(t=17.29,P<0.0001);Western blot结果表明,两组huh-7细胞蛋白表达量均明显下降(AncBE4max:t=5.57,P<0.01;Td-CGBE-NG:t=4.912,P<0.01)。结论:Td-CGBE-NG可以有效敲除huh-7肝癌细胞系的PCSK9基因,抑制该基因mRNA和蛋白的表达,为后续Td-CGBE-NG的应用奠定基础。

圣草酚通过miR-128-3p/MAPK轴对乳腺癌细胞增殖、凋亡的影响

目的圣草酚是一种重要的类黄酮,普遍存在于植物界,但少有对圣草酚的抗癌活性的报道。该研究旨在探究其对人乳腺癌(breast cancer,BC)的抗癌潜力及可能的机制。方法体外培养人乳腺癌细胞MCF-7和人乳腺上皮细胞FR2,通过MTT法检测圣草酚诱导的初始细胞毒性。再次培养MCF-7细胞,MTT法和集落形成实验评估圣草酚对MCF-7细胞增殖的影响;流式细胞术评估圣草酚对MCF-7细胞凋亡和线粒体膜电位(mitochondrial membrane potential,MMP)的作用;RT-qPCR和Western Blot法检测miR-128-3p和MAPK相关基因(p38 MAPK和HSP27)在圣草酚发挥抗癌功能中的作用。裸鼠体内异种移植模型中分析圣草酚对肿瘤生长的作用,TUNEL法检测圣草酚对细胞凋亡的影响。结果当圣草酚浓度超过12.5μmol·L^(-1)时,MCF-7细胞活力随着圣草酚浓度的增高而逐渐降低(P<0.05);圣草酚干预后,MCF-7细胞活力、集落形成数量和MMP均下调,细胞凋亡率增高(P<0.05);细胞中miR-128-3p水平增高,p38 MAPK和HSP27磷酸化水平均降低(P<0.05)。转染miR-128-3p抑制物在一定程度上可以逆转圣草酚对MCF-7细胞的影响(P<0.05);在转染miR-128-3p抑制物的基础上添加MAPK通路抑制剂可以削弱这种逆转作用(P<0.05)。此外,裸鼠体内异种移植模型实验证明了圣草酚可呈剂量依赖性降低裸鼠肿瘤组织生长,促进细胞凋亡(P<0.05)。结论圣草酚可有效抑制人乳腺癌细胞增殖并诱导细胞凋亡,这可能与其调控miR-128-3p/MAPK轴有关。

鸡骨骼肌卫星细胞系的建立及分析

旨在通过慢病毒包装SV 40-LT基因并转导PMSCs以构建鸡骨骼肌卫星细胞系。本研究采集20~30枚15胚龄健康AA鸡的胸部组织,采用混合酶消化法分离培养PMSCs。将细胞随机分为两组,每组3个重复,处理组选择SV 40-LT慢病毒转染48 h后,更换含1μg·mL^(-1)的嘌呤霉素筛选培养基于处理组和对照组(病毒不转染)。待对照组细胞全部死亡,对处理组细胞进行不断传代培养,最终获得鸡骨骼肌卫星细胞系。免疫荧光试验分析鸡骨骼肌卫星细胞标志基因PAX7的表达;采用CCK-8和细胞周期分析检测细胞增殖特性;血清依赖性和软琼脂分析检测其恶性转化情况;构建诱导分化模型检测其诱导分化能力。结果表明,PMSCs中有92%细胞PAX 7基因呈阳性,可用于后续研究。SV 40-LT基因在鸡骨骼肌卫星细胞系(IMSCs P12)中的表达是原代鸡骨骼肌卫星细胞的15倍,且连续传代至12代后,IMSCs P12与PMSCs均呈纤维样状。IMSCs P12具有更高的增殖活性且没有发生恶性转化,随后,对IMSCs P12进行诱导分化,在其增殖至70%到诱导分化1 d这一阶段,IMSCs P12与PMSCs的PAX 7基因表达下调,而MyoD和MyHC基因表达上调。结果提示,本研究通过转染SV 40-LT基因成功培养了鸡骨骼肌卫星细胞系,其具有与原代鸡骨骼肌卫星细胞相似的特性。该细胞系的建立为研究家禽骨骼肌相关的功能基因提供了新的平台,也为SV 40-LT基因在家禽细胞永生化的研究奠定了基础。

稳定表达人ACE2的Huh-7细胞系的建立及应用

为了构建稳定表达人血管紧张素转换酶2(human angiotensin converting enzyme 2,hACE2)的Huh-7细胞系,本研究构建了不带荧光的pWPXL-neo-hACE2慢病毒载体,并利用psPAX2和pMD2.G-VSVG共同转染HEK293T细胞获得慢病毒;将包装好的慢病毒感染Huh-7细胞后,利用潮霉素B筛选获得可表达hACE2的Huh-7细胞;通过间接免疫荧光法和蛋白质印迹法检测Huh-7细胞中hACE2蛋白的表达;采用流式细胞术分析Huh-7-hACE2细胞与严重急性呼吸综合征冠状病毒2(severe acute respiratory syndrome coronavirus 2,SARSCoV-2)刺突(spike,S)蛋白受体结合结构域(receptor-binding domain,RBD)的结合情况,并用SARS-CoV-2假病毒进一步测定Huh-7-hACE2细胞对SARS-CoV-2的易感性,最后利用鉴定过的Huh-7-hACE2细胞测定SARS-CoV-2中和抗体REGN10987对假病毒的中和效价。结果显示,hACE2蛋白在Huh-7细胞中成功表达,且表达的hACE2蛋白能与SARS-CoV-2 S蛋白RBD结合;相较于Huh-7细胞,Huh-7-hACE2细胞对假病毒的易感性显著提高,且假病毒中和效价测定结果与文献报道的真病毒中和效价相似。总之,本研究成功构建了稳定表达hACE2的Huh-7细胞系,并且其能应用于新型冠状病毒感染(corona virus disease 2019,COVID-19)治疗性单抗的活性评价,是研究SARS-CoV-2的致病机制、开发抗病毒药物及疫苗的有利工具。