目的:研究子痫前期患者发病机制中相关的免疫学变化及基因背景。方法:SAP法检测血液中NK细胞数量。MTT珐检测NK细胞增殖能力。51Cr释放法测定NK细胞杀伤活性。免疫组化法检测胎盘蜕膜浸润的NK细胞数量及HLA-G蛋白表达水平。RT-PCR和real...目的:研究子痫前期患者发病机制中相关的免疫学变化及基因背景。方法:SAP法检测血液中NK细胞数量。MTT珐检测NK细胞增殖能力。51Cr释放法测定NK细胞杀伤活性。免疫组化法检测胎盘蜕膜浸润的NK细胞数量及HLA-G蛋白表达水平。RT-PCR和real time PCR法检测胎盘蜕膜组织HLA-G mRNA。PCR-SSP法检测两组HLA-DRB1等位基因频率,并分析母/儿间所携带某些HLA-DRB1等位基因配伍的频率。结果:①子痫前期患者外周血及其新生儿脐血NK细胞数均显著高于正常晚孕者及其新生儿。②子痫前期患者外周血及其新生儿脐血NK细胞增殖活性及杀伤活性均明显高于正常晚孕组(P<0.05)。③轻度与重度子痫前期组胎盘蜕膜NK细胞均多于正常晚孕组,但仅重度子痫前期组蜕膜NK细胞数量增加有统计学意义(P<0.05)。④轻度子痫前期组、重度子痫前期组和正常晚孕组之间HLA-G mRNA水平两两比较差异均无统计学意义(P>0.05)。⑤轻度子痫前期组HLA-G蛋白表达水平与正常晚孕组无显著性差异,重度子痫前期组胎盘HLA-G蛋白表达水平较正常晚孕组显著降低(P<0.05)。⑥两组孕妇和新生儿均检出13个HLA-DRB1等位基因,各等位基因频率在两组间均无明显差异。子痫前期组母不携带HLA-DRB1*04基因/儿携带DRB1*04基因、均不携带HLA-DRB1*14基因的频率显著高于正常晚孕组(P<0.05);母/儿均不携带HLA-DRB1*10基因、均携带HLA-DRB1*14的频率显著低于正常晚孕组(P<0.05)。结论:①先兆子痫患者外周血、脐血及蜕膜的NK细胞数量增多,活性增强,在妊娠过程中影响母胎免疫耐受。②母婴所携带的某些HLA-DRB1基因配伍与子痫前期易感性或抗性相关;父源性HLA-DRB1*04基因与子痫前期易感性相关。展开更多
The non-classical HLA class I antigen HLA-G is an immune modulator which inhibits the functions of T cells, NK cells, and the Dendritic cells (DC). As a result, HLA-G expression in malignant cells may provide them wit...The non-classical HLA class I antigen HLA-G is an immune modulator which inhibits the functions of T cells, NK cells, and the Dendritic cells (DC). As a result, HLA-G expression in malignant cells may provide them with a mechanism to escape the immune surveillance. In melanoma, HLA-G antigen expression has been found in 30% of surgically removed lesions but in less than 1% of established cell lines. One possible mechanism underlying the differential HLA-G expression in vivo and in vitro is that the HLA-G gene is epigenetically repressed in melanoma cells in vitro. To test this hypothesis, we treated the HLA-G negative melanoma cell line OCM-1A with the DNA methyltransferase inhibitor 5-aza-2’-deoXycytidine (5-AC) and analyzed whether HLA-G expression can be restored. Our data strongly suggest that HLA-G is silenced as a result of CpG hypermethylation within a 5’ regulatory region encompassing 220 bp upstream of the start codon. After treatment, HLA-G mRNA expression was dramatically increased. Western blot and flow cytometry showed that HLA-G protein was induced. Interestingly, HLA-G cell surface expression on the 5-AC treated OCM-1 A cells is much less than that on the HLA-G positive JEG-3 cells while a similar amount of total HLA-G was observed. Possible mechanisms for the difference were analyzed in the study such as cell cold-treatment, peptide loading and antigen processing machinery components (APM) as well as β2 microglobulin (β2-m) expression. Data revealed that the APM component calreticulin might be involved in the lower HLA-G surface expression on OCM-1 A cells. Taken together, our results indicated that DNA methylation is an important epigenetic mechanism by which HLA-G antigen expression is modulated in melanoma cells in vitro. Furthermore, to the first time, we hypothesized that the deficiency of calreticulin might be involved in the low HLA-G surface expression on the 5-AC treated OCM-1 A cells.展开更多
Objective:This study aimed to evaluate the effects of acupuncture on embryo implantation and maternal–fetal immune tolerance in rats with ovarian hyperstimulation,identify the mechanisms of acupuncture treatment,and ...Objective:This study aimed to evaluate the effects of acupuncture on embryo implantation and maternal–fetal immune tolerance in rats with ovarian hyperstimulation,identify the mechanisms of acupuncture treatment,and promote application of acupuncture in the field of assisted reproduction.Methods:Eighty female Wistar rats were randomized into normal(N),model(M),acupuncture treatment(A),and progesterone treatment(P)groups,with 20 rats in each group.An ovarian hyperstimulation model was established in groups M,A,and P by the peritoneal injection of pregnant mare serum gonadotropin(PMSG)combined with human chorionic gonadotropin(HCG).Bilateral“Sanyinjiao(SP6)”,“Zusanli(ST36)”and“Taichong(LR3)”as acupoints were needled in rats in group A.Rats in group P received an intramuscular injection of progesterone(4 mg/kg).All pregnant rats were treated for seven days.On the 8th day,the pregnant rats were sacrificed for testing.The expression levels of estrogen(E2)and progesterone(P4)in serum and their receptors in the endometrium,Th1 and Th2 cytokines in serum and endometrium,RT-BM1(the counterpart of HLA-G in rats)mRNA,Ly49 activating receptors(Ly49s3,Ly49s5)and inhibitory receptors(Ly49i3,Ly49i4,Ly49i5)on immunocytes in the maternal–fetal interface were detected using ELISA,Western blot,and real-time polymerase chain reaction(PCR).Results:Compared with group N,the pregnancy rate and the number of implanted embryos were significantly decreased in group M(P<0.05);E2 and P4 levels in serum,and the protein levels of ER-αand Progesterone Receptor(PGR)in the implantation site of endometrial tissue were significantly reduced in group M(P<0.05);the expression of INF-γand IL-2 in serum and endometrium were significantly increased,while the expression of IL-4,IL-6,and IL-10 in serum and endometrium were significantly decreased in group M(P<0.05).The mRNA levels of RT-BM1,Ly49i3,and Ly49i4 receptors in group M were significantly higher than those in group N,whereas the mRNA levels of Ly49i5 and Ly49s5 receptors in group M were significantly lower than those in group N at the maternal–fetal interface(P<0.05).The pregnancy rate and number of implanted embryos were significantly higher in group A than in group M(P<0.05).Serum E2 and P4 levels were significantly higher in groups A and P than in group M(P<0.05).The expression of ER-αand PGR in the implantation site of endometrial tissue in group A were significantly higher than those in group M(P<0.05).The serum levels of INF-γand IL-2 in group A and group P were significantly lower than those in group M(P<0.05).The serum expression of IL-4,IL-6,IL-10 in group A and group P were significantly higher than those in group M(P<0.05).The endometrium expression of INF-γand IL-2 in group A were significantly lower than those in group M(P<0.05).The endometrium expression of IL-4,IL-6,IL-10 in group A were significantly higher than those in group M(P<0.05).The endometrium expression of INF-γin group P were significantly lower than those in group M(P<0.05).The endometrium expression of IL-6 and IL-10 in group P were significantly higher than those in group M(P<0.05).The mRNA levels of RT-BM1 and these Ly49 receptors(except the Ly49s3 receptor)at the maternal–fetal interface of groups A and P were higher than those of group M(P<0.05).展开更多
目的建立表达可溶性 HL A- G1蛋白的真核表达载体 pc DNA3- s HL A- G1。方法从绒癌细胞系 Jeg- 3细胞中提取总 RNA,借助 RT- PCR技术扩增可溶性 HL A - G1的 c DNA并把其插入真核表达载体 pc DNA 3,然后经酶切和测序法鉴定。结果经酶...目的建立表达可溶性 HL A- G1蛋白的真核表达载体 pc DNA3- s HL A- G1。方法从绒癌细胞系 Jeg- 3细胞中提取总 RNA,借助 RT- PCR技术扩增可溶性 HL A - G1的 c DNA并把其插入真核表达载体 pc DNA 3,然后经酶切和测序法鉴定。结果经酶切鉴定及测序分析 ,证实已成功构建 pc DNA3- s HL A- G1。结论本研究成功构建可溶性 HL A -展开更多
文摘目的:研究子痫前期患者发病机制中相关的免疫学变化及基因背景。方法:SAP法检测血液中NK细胞数量。MTT珐检测NK细胞增殖能力。51Cr释放法测定NK细胞杀伤活性。免疫组化法检测胎盘蜕膜浸润的NK细胞数量及HLA-G蛋白表达水平。RT-PCR和real time PCR法检测胎盘蜕膜组织HLA-G mRNA。PCR-SSP法检测两组HLA-DRB1等位基因频率,并分析母/儿间所携带某些HLA-DRB1等位基因配伍的频率。结果:①子痫前期患者外周血及其新生儿脐血NK细胞数均显著高于正常晚孕者及其新生儿。②子痫前期患者外周血及其新生儿脐血NK细胞增殖活性及杀伤活性均明显高于正常晚孕组(P<0.05)。③轻度与重度子痫前期组胎盘蜕膜NK细胞均多于正常晚孕组,但仅重度子痫前期组蜕膜NK细胞数量增加有统计学意义(P<0.05)。④轻度子痫前期组、重度子痫前期组和正常晚孕组之间HLA-G mRNA水平两两比较差异均无统计学意义(P>0.05)。⑤轻度子痫前期组HLA-G蛋白表达水平与正常晚孕组无显著性差异,重度子痫前期组胎盘HLA-G蛋白表达水平较正常晚孕组显著降低(P<0.05)。⑥两组孕妇和新生儿均检出13个HLA-DRB1等位基因,各等位基因频率在两组间均无明显差异。子痫前期组母不携带HLA-DRB1*04基因/儿携带DRB1*04基因、均不携带HLA-DRB1*14基因的频率显著高于正常晚孕组(P<0.05);母/儿均不携带HLA-DRB1*10基因、均携带HLA-DRB1*14的频率显著低于正常晚孕组(P<0.05)。结论:①先兆子痫患者外周血、脐血及蜕膜的NK细胞数量增多,活性增强,在妊娠过程中影响母胎免疫耐受。②母婴所携带的某些HLA-DRB1基因配伍与子痫前期易感性或抗性相关;父源性HLA-DRB1*04基因与子痫前期易感性相关。
文摘The non-classical HLA class I antigen HLA-G is an immune modulator which inhibits the functions of T cells, NK cells, and the Dendritic cells (DC). As a result, HLA-G expression in malignant cells may provide them with a mechanism to escape the immune surveillance. In melanoma, HLA-G antigen expression has been found in 30% of surgically removed lesions but in less than 1% of established cell lines. One possible mechanism underlying the differential HLA-G expression in vivo and in vitro is that the HLA-G gene is epigenetically repressed in melanoma cells in vitro. To test this hypothesis, we treated the HLA-G negative melanoma cell line OCM-1A with the DNA methyltransferase inhibitor 5-aza-2’-deoXycytidine (5-AC) and analyzed whether HLA-G expression can be restored. Our data strongly suggest that HLA-G is silenced as a result of CpG hypermethylation within a 5’ regulatory region encompassing 220 bp upstream of the start codon. After treatment, HLA-G mRNA expression was dramatically increased. Western blot and flow cytometry showed that HLA-G protein was induced. Interestingly, HLA-G cell surface expression on the 5-AC treated OCM-1 A cells is much less than that on the HLA-G positive JEG-3 cells while a similar amount of total HLA-G was observed. Possible mechanisms for the difference were analyzed in the study such as cell cold-treatment, peptide loading and antigen processing machinery components (APM) as well as β2 microglobulin (β2-m) expression. Data revealed that the APM component calreticulin might be involved in the lower HLA-G surface expression on OCM-1 A cells. Taken together, our results indicated that DNA methylation is an important epigenetic mechanism by which HLA-G antigen expression is modulated in melanoma cells in vitro. Furthermore, to the first time, we hypothesized that the deficiency of calreticulin might be involved in the low HLA-G surface expression on the 5-AC treated OCM-1 A cells.
基金Supported by grants from National Natural Science Foundation of China:81202757,81874388Hubei Provincial Traditional Chinese and Western Medicine Clinical Collaboration Program on Major Diseases(Infertility)(2023):EWBT 2023-2。
文摘Objective:This study aimed to evaluate the effects of acupuncture on embryo implantation and maternal–fetal immune tolerance in rats with ovarian hyperstimulation,identify the mechanisms of acupuncture treatment,and promote application of acupuncture in the field of assisted reproduction.Methods:Eighty female Wistar rats were randomized into normal(N),model(M),acupuncture treatment(A),and progesterone treatment(P)groups,with 20 rats in each group.An ovarian hyperstimulation model was established in groups M,A,and P by the peritoneal injection of pregnant mare serum gonadotropin(PMSG)combined with human chorionic gonadotropin(HCG).Bilateral“Sanyinjiao(SP6)”,“Zusanli(ST36)”and“Taichong(LR3)”as acupoints were needled in rats in group A.Rats in group P received an intramuscular injection of progesterone(4 mg/kg).All pregnant rats were treated for seven days.On the 8th day,the pregnant rats were sacrificed for testing.The expression levels of estrogen(E2)and progesterone(P4)in serum and their receptors in the endometrium,Th1 and Th2 cytokines in serum and endometrium,RT-BM1(the counterpart of HLA-G in rats)mRNA,Ly49 activating receptors(Ly49s3,Ly49s5)and inhibitory receptors(Ly49i3,Ly49i4,Ly49i5)on immunocytes in the maternal–fetal interface were detected using ELISA,Western blot,and real-time polymerase chain reaction(PCR).Results:Compared with group N,the pregnancy rate and the number of implanted embryos were significantly decreased in group M(P<0.05);E2 and P4 levels in serum,and the protein levels of ER-αand Progesterone Receptor(PGR)in the implantation site of endometrial tissue were significantly reduced in group M(P<0.05);the expression of INF-γand IL-2 in serum and endometrium were significantly increased,while the expression of IL-4,IL-6,and IL-10 in serum and endometrium were significantly decreased in group M(P<0.05).The mRNA levels of RT-BM1,Ly49i3,and Ly49i4 receptors in group M were significantly higher than those in group N,whereas the mRNA levels of Ly49i5 and Ly49s5 receptors in group M were significantly lower than those in group N at the maternal–fetal interface(P<0.05).The pregnancy rate and number of implanted embryos were significantly higher in group A than in group M(P<0.05).Serum E2 and P4 levels were significantly higher in groups A and P than in group M(P<0.05).The expression of ER-αand PGR in the implantation site of endometrial tissue in group A were significantly higher than those in group M(P<0.05).The serum levels of INF-γand IL-2 in group A and group P were significantly lower than those in group M(P<0.05).The serum expression of IL-4,IL-6,IL-10 in group A and group P were significantly higher than those in group M(P<0.05).The endometrium expression of INF-γand IL-2 in group A were significantly lower than those in group M(P<0.05).The endometrium expression of IL-4,IL-6,IL-10 in group A were significantly higher than those in group M(P<0.05).The endometrium expression of INF-γin group P were significantly lower than those in group M(P<0.05).The endometrium expression of IL-6 and IL-10 in group P were significantly higher than those in group M(P<0.05).The mRNA levels of RT-BM1 and these Ly49 receptors(except the Ly49s3 receptor)at the maternal–fetal interface of groups A and P were higher than those of group M(P<0.05).
文摘目的建立表达可溶性 HL A- G1蛋白的真核表达载体 pc DNA3- s HL A- G1。方法从绒癌细胞系 Jeg- 3细胞中提取总 RNA,借助 RT- PCR技术扩增可溶性 HL A - G1的 c DNA并把其插入真核表达载体 pc DNA 3,然后经酶切和测序法鉴定。结果经酶切鉴定及测序分析 ,证实已成功构建 pc DNA3- s HL A- G1。结论本研究成功构建可溶性 HL A -
