目的 研究雄性高促性腺激素性性腺功能减退症的机制,构建一种雄性高促性腺激素性性腺功能减退症小鼠模型.方法 将20只ICR雄性小鼠分为3组,对照组6只,实验组分3 mg·kg-1×10d组7只,4 mg·kg-1×10d组7只.对照组给予二甲基亚砜与生理盐水等量混合后腹腔注射,实验组给予白消安溶于二甲基亚砜与生理盐水等量混合液后腹腔注射,连续注射10d.给药40d后,取眼球血采用ELISA法检测血清性激素水平,取双侧睾丸称重,右侧睾丸分析精子数量及活力,左侧睾丸苏木精-伊红染色检测睾丸病理状态、睾丸病理评分、曲细精管直径和生精上皮厚度.结果 与对照组相比,4 mg·kg-1×10d组小鼠睾丸系数降低(P＜0.05),体质量差异无统计学意义(P＞0.05),证明此造模方法不会影响小鼠正常生长及发育.4 mg·kg-1×10d组睾丸Johnson评分低于对照组(P＜0.05).4 mg·kg-1×10d组精子数量低于对照组且低于3 mg·kg-1×10d组(P均＜0.05),4 mg·kg-1×10d组多为Ⅳ级精子,活动能力极差.4 mg·kg-1×10d组曲细精管直径和生精上皮厚度低于对照组(P均＜0.05).4 mg·kg-1×10d组卵泡刺激素(FSH)、黄体生成素(LH)浓度高于对照组(P均＜0.05),同时4 mg·kg-1×10d组睾酮浓度低于对照组(P＜0.05).睾丸HE染色显示4 mg·kg-1×10d组小鼠生精功能严重障碍,生精小管散在分布于间质中,间质细胞减少,生精小管上皮内细胞层数较少,各级生精细胞数量较少,管腔空洞,无成熟精子.结论 通过白消安小剂量持续刺激法腹腔注射成功构建了雄性高促性腺激素性性腺功能减退症小鼠模型.

During the professional study period, the professional postgraduates of reproductive endocrinology and their tutors formed an exploratory learning community. Guided by the principle of natural ecology, the tutor group taught the postgraduates interdisciplinary knowledge according to the professional characteristics during their rotation, focusing on academic issues, explaining medical classics, integrating medical theories and confirming clinical practice from the three dimensions of literature, medical science and clinical practice, and the innovative practice of medical literature integration is carried out in classroom teaching. The subtasks are extended from the opening report of postgraduates, giving full play to the flexibility of the tutor responsibility system, and absorbing postgraduates to participate in different types of sub projects and training processes. Then, taking the subtask as the carrier, the training content and practice mode of postgraduates are innovated, and various forms of academic achievements are produced step by step, continuously stimulate postgraduates’ interest in scientific research and enhance postgraduates sense of academic achievement and honor, and gradually guide postgraduates academic innovation ability and scientific research thinking, so as to achieve synchronous improvement in the three aspects of tutors’ teaching practice experience-postgraduates’ research and innovation level-talent training quality connotation.

目的 探讨宁夏及其周边地区不同睾丸生精功能患者血清中抑制素-B(inhibin-B,INHB)水平,进一步明确其与患者年龄、睾丸体积、血清性激素的关系,并分析INHB对睾丸生精功能的评估价值.方法 收集2019年1月至2020年12月就诊于宁夏医科大学总医院生殖医学中心的不孕不育患者1 730例,依据精液检查结果及手术取精结局分组,分为正常精子组1 493例、轻度少精子组55例、中度少精子组14例、重度少精子组52例、梗阻性无精子症组51例、非梗阻性无精子症组65例.比较各组睾丸体积及血清INHB、卵泡刺激素(follicle stimulating hormone,FSH)、黄体生成素(luteinizing hormone,LH)、睾酮(testosterone,T)水平;采用折线图分析患者年龄与INHB的关系;采用Pearson法分析患者睾丸体积及血清性激素水平与INHB的相关性;采用受试者工作特征(receiver operating characteristic curve,ROC)曲线分析血清FSH、INHB及睾丸体积对睾丸生精功能评估的意义.结果 各组左侧和右侧睾丸体积、血清INHB、FSH、LH、T水平比较,差异有统计学意义(P＜0.05).正常精子组和少精子组患者年龄与血清INHB水平均呈倒U型分布,31～40岁间血清INHB水平达峰后随着年龄增长而呈下降趋势.所有患者左右侧睾丸体积与血清INHB水平均呈正相关性(P＜0.05);正常精子组和少精子组患者血清FSH、LH、T与INHB水平均呈正相关性(P＜0.05).血清FSH、INHB及睾丸体积联合评估睾丸生精功能的曲线下面积为0.908,灵敏度为70.4％,特异度为92.5％.结论 睾丸体积及血清INHB、FSH三项指标对于评估睾丸生精功能均有意义,三者联合的评估价值更高.

目的 构建大鼠micro RNA-133a-3P(miR-133a-3p)过表达及干扰慢病毒载体,筛选建立稳定表达miR-133a-3p的阴茎海绵体平滑肌细胞(CCSMC)稳转细胞株.方法 原代及传代培养CCSMCs,并进行细胞免疫荧光染色鉴定.PCR技术扩增相应目的基因片段并进行酶切,回收后与目的基因连接,产物转化细菌感受态细胞,对阳性克隆测序行对比分析后,使用三质粒包装系统将含有目的基因的重组质粒和辅助包装质粒共转染至293T细胞进行慢病毒包装及纯化,并用荧光法测定病毒滴度.设置miR-133a-3p过表达组、miR-133a-3p干扰组、阴性对照组和空白对照组,在感染复数为10的条件下感染CCSMCs.倒置荧光显微镜下观察各组绿色荧光蛋白的表达情况,浓度为2μg·mL-1嘌呤霉素筛选出稳定表达miR-133a-3p的CCSMCs.qRT-PCR检测慢病毒转染后CCSMCs内miR-133a-3p的表达.结果 原代培养的大鼠CCSMCsα-SMA阳性细胞率达90％以上.经测序分析证实重组慢病毒构建正确;过表达及干扰慢病毒滴度分别为3×108和1×109 TU·mL-1;qRT-PCR结果显示,与阴性对照组相比,miR-133a-3p过表达组miR-133a-3p的相对表达量增高,miR-133a-3p干扰组miR-133a-3p的相对表达量降低(P均<0.01).结论 本实验成功构建了miR-133a-3p过表达及干扰慢病毒表达载体,对大鼠CCSMCs进行转染和筛选后,可快速、高效、低成本获得过表达及干扰miR-133a-3p的稳转株.

目前,医学研究生培养的内容以项目实验研究或基地的规范化培训为主,但明显忽视了医学人文素质内涵的培养.文章在现有研究生教育的基础上,增加导师人文素质修养的沉淀,拓展研究生人文素质教育的方法与途径,指导学生在专业理论知识的实践载体中全程化提升自身素质.导师与学生在教学—科研—学习的互动过程中,同步完成知识的传授—创新—传承,培养了研究生的人文素质和科研创新思维.

Objective To construct PRL-3 gene-specific shRNA lentivirus interference vector and infect SW480 colon cancer cells, and study the expression inhibition of PRL-3 gene in colon cancer cell lines.Methods Three interfering sequences shRNAs targeting PRL-3 were synthesized and constructed the lentiviral vector, and the recombinant was identificated by DNA sequencing analysis, then the lentiviral interference vectors of PRL-3 shRNAs were transfected and packaged into 293 T cells. The transfection efficiency was detected under fluorescence microscope. The lentiviral particles were collected to infect human colon cancer SW480 cells. The expression of PRL-3 m RNA was detected by real-time quantitative polymerase chain reaction (RT-PCR). Results DNA sequencing identified successful construction of the three PRL-3 shRNA lentivirus vectors. The recombinant lentivirus gained from 293 T cells had a titer 5×108 TU·m L-1.three PRL-3 shRNA lentivirus vectors infect SW480 cells, real-time PCR demonstrated that the interference efficiency of PRL-3 m RNA were 89.6％, 83.2％, 88.8％, respectively. PRL-3 shRNA1 showed the highest interference efficiency.Conclusion In this study, shRNA lentiviral vector targeting PRL-3 gene was successfully constructed, and PRL-3 shRNA1 is shown to inhibit the expression level of PRL-3 gene in SW480 cells.

目的 研究氧化应激及精子DNA损伤对反复早期妊娠丢失(REPL)的影响.方法 选择宁夏医科大学总医院生殖医学中心门诊收治的REPL患者46例设为REPL组,已正常生育过的男性体检者共50例作为对照组,留取丈夫精液标本,采用计算机辅助精液分析系统(CASA)检测精液常规参数,Diff-Quik试剂盒检测精子形态,通过硫代巴比妥酸法测定丙二醛(MDA)含量,采用DNA碎片率(DFI)检测试剂盒测定DNA损伤.结果 REPL组正常精子形态明显低于对照组[(4.31±0.62)％vs.(7.82 +1.21)％,P＜0.01],其中头部畸形率显著增加(50.12％ vs.44.53％);精子碎片指数显著高于对照组(28.40％ vs.16.72％,P＜0.01),MDA含量显著高于对照组[(5.67±0.74)％vs.(2.98±0.64)％,P＜0.01];REPL组DFI与精子形态及MDA水平具有显著相关性(r=0.43,P＜0.05).结论 对于REPL患者开展氧化应激水平MDA及DFI测定,对其病因诊断及治疗具有重要的临床意义.

Objective To investigate the different expression and clinical significance of peripheral blood CD8+T cells CD28 and CD57 in gastric cancer patients. Methods Peripheral blood T lymphocyte surface CD8+CD28+and CD8+CD57+ expression were detected in 66 cases of patients with gastric cancer and 23 healthy controls using flow cytometry. Results The level of peripheral blood CD8+CD28+ T cell percentage in preoperative gastric cancer patients was significantly lower than those in healthy controls(P=0.035). There was no statistically significant difference for the peripheral blood T lymphocyte CD8+CD28+ and CD8+CD57+ percentages in pre and post operation patients with gastric cancer(P>0.05). The percentages of CD8+CD57+ T cells in patients with different clinical stages showed a statistical difference(P=0.017). The percentage of CD8+CD57+T cells is lover in gastric cancer patients with lymph node metastasis group than that of withant metastasis group(P=0.051). The percentages of CD8+CD28+ T cells and CD8+CD57+ T cells was significantly higher in gastric cancer patients with invasion full-thickness stomach lining than that of no contamination full thickness stomach group(P≤0.05). Conclusion The loss of CD8+CD28+ T cells in peripheral blood may be associated with the occurrence of tumor. The increase of peripheral blood CD8+CD57+ T cells may promote tumor local invasion.

目的 探讨荧光原位杂交(FISH)技术快速诊断唐氏综合征(DS)的临床应用价值.方法 选取疑似唐氏综合征患者117例,采集外周血,分别进行染色体核型分析和间期细胞FISH试验,分析两种方法的检测结果.结果 ①疑似唐氏综合征(21-三体)患者117例,染色体核型分析与FISH试验结果均显示21-三体99例、正常核型18例,两种方法检测染色体数目异常的结果一致.②99例唐氏综合征患者中,染色体核型分析显示单纯型21-三体94例、易位型21-三体3例、嵌合型21-三体2例,FISH显示单纯型21-三体97例、嵌合型21-三体2例.结论 FISH技术适宜于染色体数目异常、不宜用于染色体结构异常的诊断,作为DS的快速诊断方法,可以在基层推广应用,对控制和降低边远地区的出生缺陷、提高人口素质具有重要意义.

目的 通过对一个三代视网膜色素变性(RP)家系进行基因检测分析,找到其致病基因.方法 采用外显子捕获直接测序,检测家系成员40个RP相关基因,经与美国国立生物技术信息中心(NCBI)的SNP数据库(dbSNP)和国际人类基因组单体型图(Haplotype Map,简称HapMap)数据库进行比较,查找致病基因.结果 该家系的致病基因位于USH2A基因.该基因编码区存在2个错义突变(p.Tyr1279Asn、p.Cys934Trp).结论 本研究在一个三代RP家系中发现了USH2A基因p.Tyr1279Asn、p.Cys934Trp两个新突变位点.

Objective To analyze chromosome karyotype of amniotic fluid of pregnant women at the second trimester of pregnancy in prenatal diagnosis and to explore the clinical significance of karyotype analysis of amniotic fluid cells in prenatal diagnosis of chromosome abnormality disease.Methods 338 cases of pregnant women at the second trimester of pregnancy with the indications of prenatal diagnosis were implemented for amniocentesis,then amniotic fluid were cultured and performed the G-banding staining for karyotype analysis.Results The success rate of amniotic fluid cells culture was 100% in 338 cases of amniotic fluid specimen.36 cases of normal variation of chromosome were found(10.65%) and 13 cases of abnormal chromosome were found(3.85%) which included 9 of numerical abnormalities chromosome(69.23% in abnormal chromosome) and 4 of structural abnormalities chromosome(30.77% in abnormal chromosome) Conclusion To apply chromosomal karyotype analysis in amniotic fluid cells and prenatal diagnosis for the pregnant women with indications of prenatal diagnosis.can be safe and effective for fetal chromosome abnormalities in prenatal diagnosis.

To investigate mutations of BRCA1/BRCA2 in familial breast cancer from Ningxia Autonomous Region.Seven families with familial breast cancer were enrolled into this study.Thirty-two samples were collected from members of the 7 families,including one cancer patient from each of the 7 families,and 25 females first degree relatives without cancer.Twenty unrelated female donors served as controls..DNAs were extracted from peripheral blood leukocytes and exon 5、11、18、20、24 of BCRA1 and exon 10、11 of BCRA2 genes were amplified by PCR and subject to direct sequencing.Eleven mutations were found in BCRA1 and all occurred in exon 11,whereas 8 mutations were found in BCRA2 gene and within exon 10.Among the 19 mutations observed,2 were found for the first time.These results suggest that Hui familial breast cancer in Ningxia Autonomous Region has its characteristic mutation pattern and may be useful to establish gene screening model for breast cancer in the region.

Objective To study the killing effect of gastric MKN-28 cells of cytokine-induced killer cells(CIK) co-cultured with dendritic cells(DC).Methods Peripheral blood mononuclear cells(PBMC) were isolated from healthy human,DC and CIK cells were induced by different cytokines,and they were co-cultured to produce DC-CIK cells.The killing activity of different CIK groups against MKN-28 was determined by MTS.Results Compared with CIK,the DC-CIK presented a significantly higher proliferation(P0.05).The killing effects of gastric MKN-28 cells were enhanced(P0.05).The activity of cytotoxicity to MKN-28 in DC-CIK group was 38.02%±2.06%,and it was stronger than that in CIK group(29.78±1.84%),respectively(E/T=20:1).Conclusion DC-CIK cell is a kind of immune cell which has a higher proliferation and cytotoxicity than that in CIK cell.

Objective To study the killing effect of dendritic cells(DC) and cytokine-induced killer(CIK) cells pulsed with specific antigens against gastric cancer cells.Methods Peripheral blood mononuclear cells(PBMC)were isolated from healthy subjects.DC and CIK cells were induced by different cytokines.Gastric cell line MKN-28 lysates of heat shock were used as antigen and pulsed DC,which co-cultured with CIK cells.The phenotypes of DC and CIK cell membranes were determined by flow cytometry,and the killing activity of CIK against MKN-28 was determined by(MTS).Results Compared with CIK,the co-cultured DC-CIK presented a significantly higher proliferation.The numbers of CD3+CD8+ and CD3+CD56+ cells increased,and the killing effect was enhanced.The cytotoxic activity to MKN-28 of DC-CIK pulsed with MKN-28 antigen(Ag-DC-CIK) group was(57.96±2.23)%,which was more strong than(38.02±2.06)% of DC-CIK pulsed without MKN-28 antigen group and(29.78±1.84)% of CIK group,respectively [the ratio of effect to target =20.0∶ 1].Conclusions Heat shock tumor-lysate-pulsed DC can strengthen the proliferation and killing activity of CIK against gastric cancer cells.

Objective To investigate the association between CYP27B1 gene promoter polymorphisms and patients with Graves' disease in Ningxia Han People.Methods A case control study was performed for CYP27B1(1260 C/A) in the promoter region in 99 patients with GD and 83 healthy individuals.The CYP27B1 genotype all cases and control were examined of by polymerase chain reaction restriction fragment length polymorphism(PCR-RFLP).Results In the promoter(1260) of CYP27B1 gene,the frequencies of genotypes and C/A allele showed no significant difference between cases and controls(P0.05).Conclusion The position 1260 C/A in the promoter of CYP27B1 may be not susceptible to GD in Ningxia Han people.

Objective To detect the prevalence of mutational sites of breast cancer susceptibility gene 1/2(BRCA1 and BRCA2) in the patients of Hui nationality with breast cancer in Ningxia area.Methods Paraffin imbedding and frozen tissues of breast cancer were collected from 60 breast cancer patients of Hui nationality and 15 cases benign breast diseases.The mutations of three exons(2,11 and 20) of BRCA1 gene and one exon(11) of BRCA2 gene were examined by polymerase chain reaction(PCR) and DNA direct sequencing.Results The mutations of BRCA1 gene were found in 10 of 60 cases(16.67%) of breast caner.No mutation was identified in the controls.There was a significant difference of the mutation rate of BRCA1 gene between lymph node metastasis and non-lymph node metastasis groups(P0.05).Conclusion BRCA1 mutation may play an important role in the risk factors of breast cancer patients of Hui nationality in Ningxia.

To investigate the killing effect of cytokine-induced killer(CIK) loaded with cell associated antigen on MAD-MB231 cell line,the peripheral blood mononuclear cells(PBMC) were obtained by density gradient centrifugation method,while the dendritic cells(DC) and CIK cells were induced by routine methods.MTT was used to detect the killing effect of three groups of cell proportions CIK,DC+CIK,antigen loaded DC+CIK MAD-MB231 under 4 different effector-target ratios.It was found that the number of CD3+/CD56+ double-expressed cella was significantly elevated when the CIK cells were co-cultivated with with DCs and DCs loaded with antigen respectively(P0.05).Also,the tumor killing activity of CIK cells was significantly increased when they were co-cultivated with DCs pulsed with antigen.These results may provide experimental basis for the immunotherapy of breast cancer.

Objective To explone the relationship between the mutation of the BRCA1 gene and the expression of ER,PR and CerbB-2 in sporadic Hui patients with breast cancer in Ningxia.Methods By using polymerase chain reaction(PCR) and DNA direct sequencing,paraffin imbedding and frozen tissues were obtained from 60 Hui female patients with breast caner and 15 controls with benign breast diseases.The expression of ER,PR and C-erbB-2 in breast cancer were analyzed retrospectively.Results 10 cases(16.67%) 60 cases with breast caner were found mutation on BRCA1.No mutation was identified in control group.The frequency of triple negative breast cancer(estrogen receptor,progesterone receptor and human epidermal growth factor receptor negative) in patients with BRCA1 mutation was 30%(3/10),which was higher than those with no BRCA1 mutation 4%,(χ2=4.364,P=0.037).There was negative correlation between the expression of ER and PR with lymph node status(χ2=5.735,P0.05;χ2=5.984,P0.05).Conclusion The mutation of BRCA1 gene were involved in carcinogensis of breast cancer.The expression of ER,PR,and C-erbB-2 has important clinical significance in treating and prognosis of breast cancer.

目的研究HR-HPV感染与宫颈癌发生发展的关系;以组织病理学诊断为金标准,评价TCT细胞学检查和HR-HPV检测在诊断CINⅡ和Ⅲ中的应用价值,为宫颈癌筛查及早期诊断提供新的检测手段。方法选取2008年1月至2009年6月在宁夏医科大学总医院就诊的宫颈病变患者159例,进行细胞学检查(TCT方法)、HR-HPV感染检测(HC-Ⅱ方法)以及组织病理学检测;依据病理学诊断,将患者分为正常、炎症、CINⅠ、CINⅡ、CINⅢ和鳞癌六组;统计学分析运用SPSS11.5软件,多个样本率的比较采用检验。结果①HR-HPV感染率在各组中差异有统计学意义(P<0.05);但在CINⅠ、Ⅱ、Ⅲ三组中差异无统计学意义(P>0.05)。②TCT细胞学筛查宫颈病变的检出率低于病理学。③HR-HPVDNA检测的灵敏度高于TCT细胞学,而后者的特异度高于前者。结论①HR-HPV的感染率随着宫颈病变程度的加重而升高,与CIN和宫颈癌的发生发存在高度一致性。②HR-HPV感染与TCT细胞学联检,可增加高级别CIN的检出率,有效预防宫颈癌的发生,在临床诊断中有重要的应用价值。

Objective To study the detection of Her2 expression in breast cancer by using immunohistochemistry and fluorescence in situ hybridization(FISH) methods,and to evaluate their clinical value.Methods The expression of Her2 protein and gene in 51 cases with breast cancer were detected by immunohistochemical staining and FISH,respectively,and then the data was analyzed.Results Of 51 cases,the expression of Her2 protein were strongly positive in 15 cases(29.41%),positive in 13 cases(25.49%),weakly positive in 14 cases(27.45%),negative in 9 cases(17.65%);Her2 gene amplification existed in 19 cases(37.25%).No significant difference was found in the results between the two methods(χ2=3.76,P0.05),but the correlation coefficient of the two methods was not very well(r=0.539,P0.01),in the(+++),(++),(+) and(-) of Her2 protein expression,the Her2 gene amplication' was 80%,23.08%,28.57% and 0,respectively.Conclusion The immunohistochemistry is regarded as the initial screening method of the detection for Her2 expression.In the cases with positive expression of Her2 protein by immunohistochemistry,it must be further detected by FISH when it selects Herceptin in clinical therapy.