BACKGROUND： Transplantation of Akt-over-expressing mesenchymal stem ceils （Akt-MSCs） has been shown to repair infarcted myocardium and improve cardiac function. However, little is known about the therapeutic effect...BACKGROUND： Transplantation of Akt-over-expressing mesenchymal stem ceils （Akt-MSCs） has been shown to repair infarcted myocardium and improve cardiac function. However, little is known about the therapeutic effects of Akt-MSCs on cardiac autonomic neuropathy in chronic heart failure （CHF）. OBJECTIVE： The present study used adriamycin-induced CHF rat models to observe the effect of Akt-MSCs on cardiac autonomic nervous regeneration and the factors mediating this effect. DESIGN, TIME AND SETTING： A randomized, controlled animal experiment was performed at the Central Laboratory of Basic Medical College, China Medical University, between September 2008 and April 2009. MATERIALS： Rabbit anti-choline acetyltransferase （CHAT）, growth associated protein-43 （GAP-43） synaptophysin （SYN） polyclonal antibodies and the secondary antibody （goat anti-rabbit IgG） were purchased from Boster, China. Cat-A-Kit assay system was provided by Amersham, USA. METHODS： （1） Adult rat MSCs were isolated and cultured for the preparation of Akt-MSCs. （2） Forty male Wistar rats were intramyocardially administered adriamycin at 2 mg/kg over 3 days for a total of five times and once a week for additional five times thereafter to establish CHF models. At 2 weeks after final adriamycin treatment, 34 successful CHF rat models were randomized to three groups： Akt-MSCs （n = 11）, simple MSCs （s-MSCs, n =11）, and control （n = 12）. Each group was intravenously administered Akt-MSCs （2x106 cells in 100 IJL PBS）, s-MSCs （2×10^6 cells in 100 μL PBS） or equal volume of phosphate buffered saline, once a day for a total of three times. MAIN OUTCOME MEASURES： At 4 weeks after final adriamycin treatment, myocardial norepinephrine （NE） content was detected using a Cat-A-Kit assay system. Myocardial CHAT, SYN and GAP-43 were performed by immunohistochemistry and Western blot analysis. Prior to, 2 and 4 weeks after adriamycin treatment, echocardiographic examination was performed and left ventricular ejection fraction （LVEF） was determined. RESULTS： Myocardial NE content, as well as SYN-positive and GAP-43-positive nerve fiber density and expression, and LVEF, was the greatest in the Akt-MSCs group, followed by the s-MSCs group, and lastly the control group （P 〈 0.05 or P 〈 0.01）. ChAT expression was similar between Akt-MSCs and s-MSCs groups, but it was higher compared with the control group （P 〈 0.05）. NE contents were negatively correlated to LVEF （r = -0.64, P = 0.015）. CONCLUSION： Transplantation of MSCs, in particular Akt-MSCs, promotes cardiac nervous regeneration in failing heart, which might be mediated by GAP-43.展开更多
Background: Protein kinase B (AKT/PKB) family is frequently amplified in ovarian cancer (OC). To the greatest of our knowledge, there is a lack of published reports about the amplification of the genes belonging to th...Background: Protein kinase B (AKT/PKB) family is frequently amplified in ovarian cancer (OC). To the greatest of our knowledge, there is a lack of published reports about the amplification of the genes belonging to the AKT family among Sudanese women with OC. The present study was conducted to detect the AKT1 gene amplification and its association with tumour types, grades, and ages among Sudanese women with OC, bearing in mind the ethnic variation. Methods: This institution-based study included 79 cases of women diagnosed with ovarian cancer (OC) at Omdurman Maternity Hospital in the period 2013-2018. Formalin-fixed, paraffin-embedded (FFPE) tissue sections were used to extract RNA. AKT1 gene amplification was assessed using quantitative real-time PCR. Results: The mean age (±SD) of included women was 49.29 (±13.612). The amplification of AKT1 gene was observed in 18/79 (22.8%) of OC women, with a high frequency in women with undifferentiated 1/2 (50%), clear cell 2/6 (33.3%), mucinous 3/11 (27.3%), endometrioid 3/17 (17.6%), and serous carcinomas 5/30 OC (16.7%). High frequency was seen in women with low (26.3%;n = 10/28) rather than in higher (19.5%;n = 8/33) grade carcinoma, and in older (25.8%;n = 8/23) rather than younger (18.2%;n = 2/9) women. No significant association between AKT1 gene amplification and tumour types, grades, and ages of women was observed (Fisher’s Exact test: p = 0.405, 0.593 and 0.851, respectively). Conclusion: AKT1 gene amplification arises in around one-fifth of Sudanese women with ovarian cancer (OC). It is seen more in undifferentiated, clear cell, and mucinous tumours types, and more frequently in low tumour grade and older women, but not to a statistically significant level. These outcomes sustenance previous studies suggesting that activated AKT genes have a vital role in OC progression and may offer a plan for targeted therapy and prognostic evaluation.展开更多
AIM:To investigate whether a virus constitutively expressing active Akt is useful to prevent cirrhosis induced by carbon tetrachloride(CCl4).METHODS:Using cre-loxp technique,we created an Ad-myr-HA-Akt virus,in which ...AIM:To investigate whether a virus constitutively expressing active Akt is useful to prevent cirrhosis induced by carbon tetrachloride(CCl4).METHODS:Using cre-loxp technique,we created an Ad-myr-HA-Akt virus,in which Akt is labeled by a HA tag and its expression is driven by myr promoter.Further,through measuring enzyme levels and histological structure,we determined the efficacy of this Ad-myrHA-Akt virus in inhibiting the development of cirrhosis induced by CCl4in rats.Lastly,using western blotting,we examined the expression levels and/or phosphorylation status of Akt,apoptotic mediators,endothelial nitric oxide synthase(eNOS),and markers for hepatic stellate cells activation to understand the underlying mechanisms of protective role of this virus.RESULTS:The Ad-myr-HA-Akt virus was confirmed using polymerase chain reaction amplification of inserted Akt gene and sequencing for full length of inserted fragment,which was consistent with the sequence reported in the GenBank.The concentrations of Admyr-HA-Akt and adenoviral enhanced green fluorescent protein(Ad-EGFP)virus used in the current study were5.5×1011vp/mL.The portal vein diameter,peak velocity of blood flow,portal blood flow and congestion index were significantly increased in untreated,saline and Ad-EGFP cirrhosis groups when compared to normal control after the virus was introduced to animal through tail veil injection.In contrast,these parameters in the Akt cirrhosis group were comparable to normal control group.Compared to the normal control,the liver function(Alanine aminotransferase,Aspartate aminotransferase and Albumin)was significantly impaired in the untreated,saline and Ad-EGFP cirrhosis groups.The Akt cirrhosis group showed significant improvement of liver function when compared to the untreated,saline and Ad-EGFP cirrhosis groups.The Hyp level and portal vein pressure in Akt cirrhosis groups were also significantly lower than other cirrhosis groups.The results of HE and Van Gieson staining indicated that Akt group has better preservation of histological structure and less fibrosis than other cirrhosis groups.The percentage of apoptotic cell was greatly less in Akt cirrhosis group than in other cirrhosis groups.Akt group showed positive HA tag and an increased level of phosphorylated Akt as well as decreased levels of Fas.In contrast,Caspase-3 and Caspase-9 levels in Akt group were significantly lower than other cirrhosis groups.Noticeable decrease of DR5 andα-SMA and increase of phosphorylated eNOS were observed in the Akt group when compared to other cirrhosis groups.The NO level in liver was significantly higher in Akt group than other cirrhosis groups,which was consistent with the level of phosphorylated eNOS in these groups.CONCLUSION:This study suggest that Ad-myr-HA-Akt virus is a useful tool to prevent CCl4-induced cirrhosis in rat model and Akt pathway may be a therapeutic target for human cirrhosis.展开更多
This study examined the role of EMP-1 in tumorigenesis of non-small cell lung carcinoma (NSCLC) and the possible mechanism. Specimens were collected from 28 patients with benign lung diseases and 28 with NSCLC, and im...This study examined the role of EMP-1 in tumorigenesis of non-small cell lung carcinoma (NSCLC) and the possible mechanism. Specimens were collected from 28 patients with benign lung diseases and 28 with NSCLC, and immunohis to chemically detected to evaluate the correlation of EMP-1 expression to the clinical features of NSCLC. Recombinant adenovirus was constructed to over-express EMP-1 and then infect PC9 cells. Cell proliferation was measured by Ki67 staining. Western blotting was performed to examine the effect of EMP-1 on the PI3K/AKT signaling. Moreover, tumor xeno-grafts were established by subcutaneous injection of PC9 cell suspension (about 5×107/mL in 100 μL of PBS) into the right hind limbs of athymic nude mice. The results showed EMP-1 was significantly up-regulated in NSCLC patients as compared with those with benign lung diseases. Over-expression of EMP-1 promoted proliferation of PC9 cells, which coincided with the activation of the PI3K/AKT pathway. EMP-1 promoted the growth of xenografts of PC9 cells in athymic nude mice. It was concluded that EMP-1 expression may contribute to the development and progress of NSCLC by activating PI3K/AKT pathway.展开更多
The reversing effect of wild-type PTEN gene on resistance of C 13K cells to cisplatin and its inhibitory effect on the phosphorylation of protein kinase B （AKT） were studied. The expression of PTEN mRNA and protein ...The reversing effect of wild-type PTEN gene on resistance of C 13K cells to cisplatin and its inhibitory effect on the phosphorylation of protein kinase B （AKT） were studied. The expression of PTEN mRNA and protein in OV2008 cells and C13K cells were semi-quantitatively detected by using RT-PCR and Western blotting. Recombinant eukaryotic expression plasmid containing human wild-type PTEN gene was transfected into C13K cells by lipofectamine2000. The expression of PTEN mRNA was monitored by RT-PCR and the expression of PTEN, Akt, p-Akt protein were ana- lyzed by Western blotting in PTEN-transfected and non-transfected C13K cells. Proliferation and chemosensitivity of cells to DDP were measured by MTT, and cell apoptosis was detected by flow cytometry after treatment with cisplatin. The expression of PTEN mRNA and protein in OV2008 cells were significantly higher than those in C13K cells. After transfection with PTEN gene for 48 h, the expression of PTEN mRNA and protein in C 13K cells were 2.04 ± 0.10, 0.94± 0.04 respectively and the expression of p-Akt protein （ 0.94± 0.07） was lower than those in control groups （1.68 ±0.14, 1.66± 0.10） （P〈 0.05）. The IC50 of DDP to C 13 K cells transfected with PTEN （7.2± 0.3 la mol/L） was obviously lower than those of empty-vector transfected cells and non-transfected cells （12.7±0.4 lamol/1, 13.0±0.3 lamol/L） （P〈0.05）. The apopototis ratio of wild-type PTEN-transfected, empty vector transfected and non-transfected C13K cells were （41.65___0.87）%, （18.61 ±0.70）% and （15.28±0.80）% respectively, and the difference was statistically significant （P〈0.05）. PTEN gene plays an important role in ovarian cancer multidrug resistance. Transfection of PTEN could increase the expression of PTEN and restore drug sensitivity to cisplatin in human ovarian cancer cell line C 13K with multidrug-resistance by decreasing the expression of p-Akt.展开更多
【目的】基于PI3K/Akt通路研究含凝血酶敏感素基序的去解联金属蛋白酶1(a disintegrin and metalloproteinase with thrombospondin motifs 1,ADAMTS1)在产双胎和单胎蒙古羊发情周期不同阶段卵巢内的表达规律,探究ADAMTS1基因影响蒙古...【目的】基于PI3K/Akt通路研究含凝血酶敏感素基序的去解联金属蛋白酶1(a disintegrin and metalloproteinase with thrombospondin motifs 1,ADAMTS1)在产双胎和单胎蒙古羊发情周期不同阶段卵巢内的表达规律,探究ADAMTS1基因影响蒙古羊繁殖性状的作用机制。【方法】选取经产2次的双胎和单胎蒙古羊共40只,自然发情,确定母羊发情期和间情期,分为双胎发情期、双胎间情期、单胎发情期和单胎间情期组,每组各10只羊。利用注射器抽取2~3岁蒙古羊颗粒细胞,分为对照组和试验组,对照组不添加药物,试验组添加15μmol/L LY294002处理颗粒细胞,分别在24、48和72 h测定细胞活力。通过实时荧光定量PCR技术检测母羊卵巢组织和颗粒细胞内ADAMTS1、PI3K、PTEN、Akt、RPS6、Bcl-2和BAX基因的相对表达量;通过Western blotting技术检测卵巢和颗粒细胞内ADAMTS1、PI3K和Akt蛋白的表达丰度。【结果】实时荧光定量PCR结果显示,双胎发情期组ADAMTS1、Bcl-2基因表达量均显著高于其余各组(P<0.05);双胎发情期组和双胎间情期组PI3K、Akt和PTEN基因表达量均显著高于单胎发情期组和单胎间情期组(P<0.05);单胎间情期组RPS6基因表达量显著低于其余各组(P<0.05);间情期与发情期相比,BAX基因表达显著上调(P<0.05)。Western blotting结果显示,双胎发情期ADAMST1蛋白表达量显著高于其余各组(P<0.05);双胎组与单胎组相比,PI3K和Akt蛋白均表达上调,其中,双胎发情期和双胎间情期PI3K蛋白表达量均显著高于单胎发情期组和单胎间情期组(P<0.05),双胎间情期Akt蛋白表达量最高,显著高于其余各组(P<0.05)。与对照组相比,试验组蒙古羊颗粒细胞形态异常,细胞增殖缓慢,且ADAMTS1、PI3K和Akt基因和蛋白的相对表达量均显著降低(P<0.05)。【结论】ADAMTS1、PI3K和Akt基因和蛋白在蒙古羊卵巢和颗粒细胞内的表达趋势一致,呈正向相关关系,证明ADAMTS1基因PI3K/Akt轴在蒙古羊产双胎性状中发挥重要作用。展开更多
目的通过研究沉默信息调节因子1(SIRT1)基因敲除对骨关节炎小鼠VEGF/AKT通路的作用,探讨骨关节炎关节软骨退变的可能机制。方法将小鼠分为两组:SIRT1^(+/+)小鼠骨关节炎模型组(A组,n=6);SIRT1^(-/-)小鼠骨关节炎模型组(B组,n=6)。荧光...目的通过研究沉默信息调节因子1(SIRT1)基因敲除对骨关节炎小鼠VEGF/AKT通路的作用,探讨骨关节炎关节软骨退变的可能机制。方法将小鼠分为两组:SIRT1^(+/+)小鼠骨关节炎模型组(A组,n=6);SIRT1^(-/-)小鼠骨关节炎模型组(B组,n=6)。荧光定量聚合酶链反应检测SIRT1基因表达情况;HE染色、番红O-固绿双染色观察膝关节软骨形态结构改变,Mankin评分评价膝关节关节软骨退变,免疫组化染色检测膝关节软骨细胞中SIRT1、VEGF和AKT蛋白水平。结果 B组SIRT1 m RNA表达量为2.24417±1.316569,明显低于A组(P<0.01)。HE染色和番红O-固绿双染色结果显示,B组膝关节关节软骨退变明显,Mankin评分分值为9.8333±1.94079分,明显高于A组(P<0.05),B组SIRT1、VEGF和AKT免疫组化染色积分分别为3.3333±1.96638、10.0000±2.44949和1.3333±1.21106,B组SIRT1蛋白表达与A组相比差异不显著(P>0.05);B组VEGF蛋白表达明显高于A组(P<0.05);B组AKT蛋白表达明显低于A组(P<0.01)。结论 SIRT1基因敲除可能通过激活VEGF/AKT通路加重骨关节炎关节软骨的退变,因此SIRT1基因可能对骨关节炎起到保护作用。展开更多
基金Scientific Research Program of Higher Education Institute in Liaoning Province, No. 2008S248
文摘BACKGROUND： Transplantation of Akt-over-expressing mesenchymal stem ceils （Akt-MSCs） has been shown to repair infarcted myocardium and improve cardiac function. However, little is known about the therapeutic effects of Akt-MSCs on cardiac autonomic neuropathy in chronic heart failure （CHF）. OBJECTIVE： The present study used adriamycin-induced CHF rat models to observe the effect of Akt-MSCs on cardiac autonomic nervous regeneration and the factors mediating this effect. DESIGN, TIME AND SETTING： A randomized, controlled animal experiment was performed at the Central Laboratory of Basic Medical College, China Medical University, between September 2008 and April 2009. MATERIALS： Rabbit anti-choline acetyltransferase （CHAT）, growth associated protein-43 （GAP-43） synaptophysin （SYN） polyclonal antibodies and the secondary antibody （goat anti-rabbit IgG） were purchased from Boster, China. Cat-A-Kit assay system was provided by Amersham, USA. METHODS： （1） Adult rat MSCs were isolated and cultured for the preparation of Akt-MSCs. （2） Forty male Wistar rats were intramyocardially administered adriamycin at 2 mg/kg over 3 days for a total of five times and once a week for additional five times thereafter to establish CHF models. At 2 weeks after final adriamycin treatment, 34 successful CHF rat models were randomized to three groups： Akt-MSCs （n = 11）, simple MSCs （s-MSCs, n =11）, and control （n = 12）. Each group was intravenously administered Akt-MSCs （2x106 cells in 100 IJL PBS）, s-MSCs （2×10^6 cells in 100 μL PBS） or equal volume of phosphate buffered saline, once a day for a total of three times. MAIN OUTCOME MEASURES： At 4 weeks after final adriamycin treatment, myocardial norepinephrine （NE） content was detected using a Cat-A-Kit assay system. Myocardial CHAT, SYN and GAP-43 were performed by immunohistochemistry and Western blot analysis. Prior to, 2 and 4 weeks after adriamycin treatment, echocardiographic examination was performed and left ventricular ejection fraction （LVEF） was determined. RESULTS： Myocardial NE content, as well as SYN-positive and GAP-43-positive nerve fiber density and expression, and LVEF, was the greatest in the Akt-MSCs group, followed by the s-MSCs group, and lastly the control group （P 〈 0.05 or P 〈 0.01）. ChAT expression was similar between Akt-MSCs and s-MSCs groups, but it was higher compared with the control group （P 〈 0.05）. NE contents were negatively correlated to LVEF （r = -0.64, P = 0.015）. CONCLUSION： Transplantation of MSCs, in particular Akt-MSCs, promotes cardiac nervous regeneration in failing heart, which might be mediated by GAP-43.
文摘Background: Protein kinase B (AKT/PKB) family is frequently amplified in ovarian cancer (OC). To the greatest of our knowledge, there is a lack of published reports about the amplification of the genes belonging to the AKT family among Sudanese women with OC. The present study was conducted to detect the AKT1 gene amplification and its association with tumour types, grades, and ages among Sudanese women with OC, bearing in mind the ethnic variation. Methods: This institution-based study included 79 cases of women diagnosed with ovarian cancer (OC) at Omdurman Maternity Hospital in the period 2013-2018. Formalin-fixed, paraffin-embedded (FFPE) tissue sections were used to extract RNA. AKT1 gene amplification was assessed using quantitative real-time PCR. Results: The mean age (±SD) of included women was 49.29 (±13.612). The amplification of AKT1 gene was observed in 18/79 (22.8%) of OC women, with a high frequency in women with undifferentiated 1/2 (50%), clear cell 2/6 (33.3%), mucinous 3/11 (27.3%), endometrioid 3/17 (17.6%), and serous carcinomas 5/30 OC (16.7%). High frequency was seen in women with low (26.3%;n = 10/28) rather than in higher (19.5%;n = 8/33) grade carcinoma, and in older (25.8%;n = 8/23) rather than younger (18.2%;n = 2/9) women. No significant association between AKT1 gene amplification and tumour types, grades, and ages of women was observed (Fisher’s Exact test: p = 0.405, 0.593 and 0.851, respectively). Conclusion: AKT1 gene amplification arises in around one-fifth of Sudanese women with ovarian cancer (OC). It is seen more in undifferentiated, clear cell, and mucinous tumours types, and more frequently in low tumour grade and older women, but not to a statistically significant level. These outcomes sustenance previous studies suggesting that activated AKT genes have a vital role in OC progression and may offer a plan for targeted therapy and prognostic evaluation.
文摘AIM:To investigate whether a virus constitutively expressing active Akt is useful to prevent cirrhosis induced by carbon tetrachloride(CCl4).METHODS:Using cre-loxp technique,we created an Ad-myr-HA-Akt virus,in which Akt is labeled by a HA tag and its expression is driven by myr promoter.Further,through measuring enzyme levels and histological structure,we determined the efficacy of this Ad-myrHA-Akt virus in inhibiting the development of cirrhosis induced by CCl4in rats.Lastly,using western blotting,we examined the expression levels and/or phosphorylation status of Akt,apoptotic mediators,endothelial nitric oxide synthase(eNOS),and markers for hepatic stellate cells activation to understand the underlying mechanisms of protective role of this virus.RESULTS:The Ad-myr-HA-Akt virus was confirmed using polymerase chain reaction amplification of inserted Akt gene and sequencing for full length of inserted fragment,which was consistent with the sequence reported in the GenBank.The concentrations of Admyr-HA-Akt and adenoviral enhanced green fluorescent protein(Ad-EGFP)virus used in the current study were5.5×1011vp/mL.The portal vein diameter,peak velocity of blood flow,portal blood flow and congestion index were significantly increased in untreated,saline and Ad-EGFP cirrhosis groups when compared to normal control after the virus was introduced to animal through tail veil injection.In contrast,these parameters in the Akt cirrhosis group were comparable to normal control group.Compared to the normal control,the liver function(Alanine aminotransferase,Aspartate aminotransferase and Albumin)was significantly impaired in the untreated,saline and Ad-EGFP cirrhosis groups.The Akt cirrhosis group showed significant improvement of liver function when compared to the untreated,saline and Ad-EGFP cirrhosis groups.The Hyp level and portal vein pressure in Akt cirrhosis groups were also significantly lower than other cirrhosis groups.The results of HE and Van Gieson staining indicated that Akt group has better preservation of histological structure and less fibrosis than other cirrhosis groups.The percentage of apoptotic cell was greatly less in Akt cirrhosis group than in other cirrhosis groups.Akt group showed positive HA tag and an increased level of phosphorylated Akt as well as decreased levels of Fas.In contrast,Caspase-3 and Caspase-9 levels in Akt group were significantly lower than other cirrhosis groups.Noticeable decrease of DR5 andα-SMA and increase of phosphorylated eNOS were observed in the Akt group when compared to other cirrhosis groups.The NO level in liver was significantly higher in Akt group than other cirrhosis groups,which was consistent with the level of phosphorylated eNOS in these groups.CONCLUSION:This study suggest that Ad-myr-HA-Akt virus is a useful tool to prevent CCl4-induced cirrhosis in rat model and Akt pathway may be a therapeutic target for human cirrhosis.
基金supported by grants from the National Natural Science Foundation of China (Nos.81072431,30872472,30973496 and 30800569)the Innovative Foundation of Huazhong University of Science and Technology (No.2010MS027)+1 种基金the Foundation of "973" Program (No.2009CB521802)by Special Fund for Central University Basic Scientific Research (Nos.2011JC062,2011JC063)
文摘This study examined the role of EMP-1 in tumorigenesis of non-small cell lung carcinoma (NSCLC) and the possible mechanism. Specimens were collected from 28 patients with benign lung diseases and 28 with NSCLC, and immunohis to chemically detected to evaluate the correlation of EMP-1 expression to the clinical features of NSCLC. Recombinant adenovirus was constructed to over-express EMP-1 and then infect PC9 cells. Cell proliferation was measured by Ki67 staining. Western blotting was performed to examine the effect of EMP-1 on the PI3K/AKT signaling. Moreover, tumor xeno-grafts were established by subcutaneous injection of PC9 cell suspension (about 5×107/mL in 100 μL of PBS) into the right hind limbs of athymic nude mice. The results showed EMP-1 was significantly up-regulated in NSCLC patients as compared with those with benign lung diseases. Over-expression of EMP-1 promoted proliferation of PC9 cells, which coincided with the activation of the PI3K/AKT pathway. EMP-1 promoted the growth of xenografts of PC9 cells in athymic nude mice. It was concluded that EMP-1 expression may contribute to the development and progress of NSCLC by activating PI3K/AKT pathway.
基金a grant from the National Natural Sciences Foundation of China (No. 30571950)National Key Basic Research Program Foundation (N0.2002CB513107).
文摘The reversing effect of wild-type PTEN gene on resistance of C 13K cells to cisplatin and its inhibitory effect on the phosphorylation of protein kinase B （AKT） were studied. The expression of PTEN mRNA and protein in OV2008 cells and C13K cells were semi-quantitatively detected by using RT-PCR and Western blotting. Recombinant eukaryotic expression plasmid containing human wild-type PTEN gene was transfected into C13K cells by lipofectamine2000. The expression of PTEN mRNA was monitored by RT-PCR and the expression of PTEN, Akt, p-Akt protein were ana- lyzed by Western blotting in PTEN-transfected and non-transfected C13K cells. Proliferation and chemosensitivity of cells to DDP were measured by MTT, and cell apoptosis was detected by flow cytometry after treatment with cisplatin. The expression of PTEN mRNA and protein in OV2008 cells were significantly higher than those in C13K cells. After transfection with PTEN gene for 48 h, the expression of PTEN mRNA and protein in C 13K cells were 2.04 ± 0.10, 0.94± 0.04 respectively and the expression of p-Akt protein （ 0.94± 0.07） was lower than those in control groups （1.68 ±0.14, 1.66± 0.10） （P〈 0.05）. The IC50 of DDP to C 13 K cells transfected with PTEN （7.2± 0.3 la mol/L） was obviously lower than those of empty-vector transfected cells and non-transfected cells （12.7±0.4 lamol/1, 13.0±0.3 lamol/L） （P〈0.05）. The apopototis ratio of wild-type PTEN-transfected, empty vector transfected and non-transfected C13K cells were （41.65___0.87）%, （18.61 ±0.70）% and （15.28±0.80）% respectively, and the difference was statistically significant （P〈0.05）. PTEN gene plays an important role in ovarian cancer multidrug resistance. Transfection of PTEN could increase the expression of PTEN and restore drug sensitivity to cisplatin in human ovarian cancer cell line C 13K with multidrug-resistance by decreasing the expression of p-Akt.
文摘目的通过研究沉默信息调节因子1(SIRT1)基因敲除对骨关节炎小鼠VEGF/AKT通路的作用,探讨骨关节炎关节软骨退变的可能机制。方法将小鼠分为两组:SIRT1^(+/+)小鼠骨关节炎模型组(A组,n=6);SIRT1^(-/-)小鼠骨关节炎模型组(B组,n=6)。荧光定量聚合酶链反应检测SIRT1基因表达情况;HE染色、番红O-固绿双染色观察膝关节软骨形态结构改变,Mankin评分评价膝关节关节软骨退变,免疫组化染色检测膝关节软骨细胞中SIRT1、VEGF和AKT蛋白水平。结果 B组SIRT1 m RNA表达量为2.24417±1.316569,明显低于A组(P<0.01)。HE染色和番红O-固绿双染色结果显示,B组膝关节关节软骨退变明显,Mankin评分分值为9.8333±1.94079分,明显高于A组(P<0.05),B组SIRT1、VEGF和AKT免疫组化染色积分分别为3.3333±1.96638、10.0000±2.44949和1.3333±1.21106,B组SIRT1蛋白表达与A组相比差异不显著(P>0.05);B组VEGF蛋白表达明显高于A组(P<0.05);B组AKT蛋白表达明显低于A组(P<0.01)。结论 SIRT1基因敲除可能通过激活VEGF/AKT通路加重骨关节炎关节软骨的退变,因此SIRT1基因可能对骨关节炎起到保护作用。
