荧光定量PCR对血及痰培养物中结核分枝杆菌DNA的检测

Objective To evaluate if fluorescence quantitative PCR (FQ-PCR) detection for Mycobacterium tuberculosis in blood or sputum culture can help tuberculosis (TB) diagnosis. Methods A total of 81 patients with a clinical diagnosis of tuberculosis but sputum negative were enrolled, 40 were tuberculosis group and 41 coexisting HIV were HIV-TB group. Blood and sputum were cultured for bacilli or L-forms of Mycobacterium tuberculosis, and FQ-PCR was used to detected bacilli DNA. Results For pulmonary tuberculosis group, 54.1 %(20/37) were positive for bacilli or Informs of Mycobacterium tuberculosis in sputum by FQ-PCR, 27.5% (11/40) were positive in blood culture, 22.5%(9/40) were positive in blood by FQ-PCR, and the total positive rate of blood was 42.5% (17/40). But for HIV-TB group, only 2 positive cultures were found in 10 sputum, the positive rate of blood culture was 7.3% (3/41), and the positive rate of blood was 17.1%(7/41) by FQ-PCR. There was no significant difference between two groups in the positive rate of Mycobacterium tuberculosis by FQ-PCR after blood cultures (P > 0.05). The total positive rates detected by FQ-PCR of sputum or blood cultures were 65.0% (26/40) and 22.0% (9/41) respectively, and there was significant difference between two groups( χ2 = 15.305, P < 0.01). Conclusions FQ-PCR for blood or sputum culture detection appears to be a useful method to diagnose TB in persons with or without HIV infection. The use of FQ-PCR significantly enhance the efficiency of the etiological diagnosis of sputum negative pulmonary tuberculosis.     

荧光定量PCR对血及痰培养物中结核分枝杆菌DNA的检测

Objective To evaluate if fluorescence quantitative PCR (FQ-PCR) detection for Mycobacterium tuberculosis in blood or sputum culture can help tuberculosis (TB) diagnosis. Methods A total of 81 patients with a clinical diagnosis of tuberculosis but sputum negative were enrolled, 40 were tuberculosis group and 41 coexisting HIV were HIV-TB group. Blood and sputum were cultured for bacilli or L-forms of Mycobacterium tuberculosis, and FQ-PCR was used to detected bacilli DNA. Results For pulmonary tuberculosis group, 54.1 %(20/37) were positive for bacilli or Informs of Mycobacterium tuberculosis in sputum by FQ-PCR, 27.5% (11/40) were positive in blood culture, 22.5%(9/40) were positive in blood by FQ-PCR, and the total positive rate of blood was 42.5% (17/40). But for HIV-TB group, only 2 positive cultures were found in 10 sputum, the positive rate of blood culture was 7.3% (3/41), and the positive rate of blood was 17.1%(7/41) by FQ-PCR. There was no significant difference between two groups in the positive rate of Mycobacterium tuberculosis by FQ-PCR after blood cultures (P > 0.05). The total positive rates detected by FQ-PCR of sputum or blood cultures were 65.0% (26/40) and 22.0% (9/41) respectively, and there was significant difference between two groups( χ2 = 15.305, P < 0.01). Conclusions FQ-PCR for blood or sputum culture detection appears to be a useful method to diagnose TB in persons with or without HIV infection. The use of FQ-PCR significantly enhance the efficiency of the etiological diagnosis of sputum negative pulmonary tuberculosis.     

软骨样副脊索瘤（附病例报告）

To study the clinical pathological characteristics and differentiation diagnosis of the chondroid parachordoma from primary tumors or metastatic to ulna, femur and radius. Methods Three cases of primary chondroid parachordoma of the ulma, femur and radius were studied by histopathological observation and immunohisto chemical staining and by pathological consultation. Results The histological features of tumor were composed of two the chordoma ceils subtotal and the chondrosareoma ceils of small - portio. The tumor was arranged vary in sizes of lobular and brink of lobular were seplum small girder of chondroid, or the tissue fiber. The tumor cells were distributed sheet, trabecula with or island -like in the intralobular, and the tumor ceils were vary in size and the karyon heterotypic were no patent and the cytoplasm of rich shows large vacuole. The chondroidosarcomas intra - lobular were viewed ceils both nucleus and uninuclear of chondrolacunae and the stroma were hyaling cartilage with or blennoid in the mesenchyma, and that picture of typical chondrosarcoma. EMA, CK, Vimentin, S - 100 protein, NSE with immunohistochemical method (ABC) staining were positive and Desmin, CD34 negative in the all of tumor cells. The chondroid parachodoma was diagnosed in the pathological read- section- conference and consulation. Conclusion Primary ulna, femur and radius chondroid parachordoma is a rare in rate of neoplasm and is diagnosed diflqculdy. Immunohistochemical method would be helpful to diagnosis.     

细菌16S rRNA在诊断非中性粒细胞性腹水自发性细菌性腹膜炎中的应用

Objective To evaluate clinical applications on the quantitative PCR detection of ascitic bacterial 16S rRNA genes in the diagnosis of spontaneous bacterial peritonitis with non-neutrophil ascites. Methods The bacterial 16S rRNA was detected by quantitative fluorescent PCR in 64 cases of suspected spontaneous bacterial peritonitis with nonneutrophil ascites, 6 cases of chronic liver disease accompanied with non-infected ascites at the same time period, as compared to the ascitic bacterial culture. Results Bacterial 16S rRNA quantitative PCR can detect DNA duplicate as low as 10 copies. The rate of detectable bacterial 16S rRNA by quantitative PCR( 15.63% ) was significantly higher than the rate of positive bacterial culture (3.13% ) ( x2 = 5.52, P ＜ 0.05 ); and 6 cases of non-infected ascites were negative analyzed by quantitative fluorescent PCR and bacterial cultures. Conclusions The ascitic bacterial 16S rRNA quantitative PCR detection is high specificity and sensitivity for the diagnosis of spontaneous bacterial peritonitis with non-neutrophils ascites, whtich is of important clinical significance.     

Diagnostic value of vascular endothelial growth factor combined with β-human chorionic gonadotropin for suspected ectopic pregnancy

Objective To explore the diagnostic values of vascular endothelial growth factor (VEGF) and p -human chorionic gonadotropin( β -hCG) for early ectopic pregnancy. Methods The serum levels of VEGF and β-hCG were measured in 109 patients with suspected ectopic pregnancy by ELISA and RIA respectively, receiver operating characteristic (ROC) curvers were established, the diagnostic values of VEGF and β -hCG for ectopic pregnancy were analyzed. Results One hundred and nine patients with suspected ectopic pregnancy were diagnosed in the end: 62 cases with ectopic pregnancy and 47 cases with intrauterine pregnancy. The levels of VEGF in ectopic pregnancy were significantly higher than those in intrauterine pregnancy (P ＜ 0.05), but the levels of β -hCG were significantly lower than those in intrauterine pregnancy (P ＜ 0.05). The areas under ROC curves of VEGF were 0.87, the areas under ROC curves of β -hCG were 0.71. In VEGF ＞ 140 ng/L for diagnosis of ectopic pregnancy, the sensitivity and specificity were 75.8% (47/62) and 80.9% (38/47) respectively. In β-hCG ＜ 1200 U/L for diagnosis of ectopic pregnancy, the sensitivity and specificity were 64.5%(40/62) and 61.7%(29/47) respectively. The sensitivity and specificity of combined detection of VEGF and β -hCG were 80.6%(50/62) and 57.4%( 27/47) respectively. Conclusions The diagnostic values of VEGF are better than those of β -hCG. The combined detection of VEGF and β -hCG can increase the rate of early diagnosis and shorten observation time.     

The immunogenicity and safety of 10 μg recombination yeast hepatitis B vaccine applied in expanded population

Objective To assess the feasibility of the 10 μg recombination yeast hepatitis B vaccine in the expanded applicable population group aged 5-18. Methods People with both HBsAg and anti-HBs negative were selected to take two-stage clinical experiment and the safety and immunogenicity were observed. Safety observation was conducted in 925 subjects, while 568 for immunogenicity. The observation group (aged 5-18) included 493 subjects, and (age>18) 75 enrolled in control group. For the observation group,there were three sub-groups including a child group (141, aged 5-6), early youth group(177, aged 12-13), and youth group (175, aged 16-18). Both groups were administered with 10 μg recombination yeast hepatitis B vaccines with 3 doses at 0 month, 1st month,6th month. To assess the immunogenicity,the vaccination reactions were observed during the following 4 weeks in order to assess the vaccine safety. The blood samples were taken during 4-6 weeks after fully vaccinated, and then anti-HBs were tested with RIA and analyzed by comparing the positive rate of anti-HBs,the geometric mean titer(GMT) and the protective rate between the two groups. Results Both observation and control group didn't show any general reactions,adverse events following immunization(AEFI) or coincidental cases when observed at 0. 5 h,6 h,24 h,48 h, 72 h, 1 week,2 weeks,3 weeks,4 weeks after being vaccinated. The result of serum test showed, the positive rates of child group, early youth group, youth group and control group were respectively 100. 00% (141/ 141),97.18% (172/177), 98. 29% (172/175) and 89. 33% (67/75); the GMTs of anti-HBs were respectively 440. 28,875.38,467.80,131.06 U/L; the protective rates were respectively 100. 00% (141/ 141), 97.18%(172/177), 97.14% (170/175) and 86. 67% (65/75). The positive rate, GMT and protective rate of the experimental group were all higher than that of control group(χ2<,positive rate>=12.77,5.12, 7.99; tGMT=3.89,4.13,5.91;χ2protective rate>=16. 81,8.60,8.44;P< 0.05). Conclusion This vaccine could be expanded to 5-18 year-old population with safety and effectiveness, the positive rate and protective rate of anti-HBs were both higher than that of control group.     

妊娠各期孕妇甲状腺激素变化的探讨

Objective To investigate the state of thyroid hormones and thyrotropin level in different gestation periods. Methods FT3, FT4, TSH and hCG were measured in 623 patients by ACS-180 automatic chemiluninescence analyzer. The urinary iodine was random measured by the use of iodine catalytic reaction mechanism of Arsenic-Cerium, rapid urinary iodine was detected with cold digestion method of quantitative detection. Result Serum FT3,FT4 ,and hCG levels in the first and second trimester were higher than in the third trimester ( F =53. 19,78.00,58.77, P ＜0. 05) ,and TSH levels were lower than in the third trimester ( F =5.68, P ＜0.05). The incidence of thyrotropin level disorder was 7. 54%, there were no significant differences in the gestation periods ( χ2 = 3.92, P ＞ 0. 05 ). The incidence of subclinical hypothyroidism,subclinical hyperthyroidism, hypothyroidism and hyperthyroidism were 3.53%, 2. 57%, 1.12% and 0. 32%, respectively. The incidence of subclinical hypothyroidism in the first and third trimester was high, while the incidence of subclinical hyperthyroidism in the second trimester was high. Conclusions Thyrotropin level in the third trimester was lower than that of the first and second trimester. HCG changes were not the main reason that caused the thyrotropin level disorder during the pregnancy.     

组氨酸脱羧酶、肠脂肪酸结合蛋白和二胺氧化酶在肠梗阻患者肠黏膜损伤诊断中的价值

Objective To investigate the clinical values of serum histidine decarboxylase(HDC),intestinal fatty acid binding protein(I-FABP),and diamine oxidase(DAO)for diagnosing intestinal mucosal injury (IMI)in patients with intestinal obstruction.Methods The expression levels of serum HDC,I-FABP,and DAO in 28 patients with strangulated intestinal obstruction,19 patients with simple intestinal obstruction,17 patients with acute simple appendicitis,and 20 healthy control were determined by enzyme-linked immunosorbent assay (ELISA)before clinical treatment,and then the areaa under receiver operating characteristic curves(AUC)of these diagnostic indicators were compared.In addition,the incidences of systemic inflammatory response syndrome (SIRS)and infectious complications were closely observed.The difference of the expressions of HDC,I-FABP,and DAO and their relationship with SIRS and infectious complications were compared among these patients and controls.Results The expression levels of serum HDC, I-FABP, and DAO were the highest in patients with strangulated intestinal obstruction (all P < 0.001), and the expression levels of these three indicators were significantly higher in patients with simple intestinal obstruction than in those with acute simple appendicitis or healthy controls (all P<0.05).The AUC of HDC (0.913) was significantly larger than that of I-FABP (0.877, P =0.000) and DAO (0.873, P = 0.000).When the cut-off value of HDC ≥31.00 ng/ml, the sensitivity, specificity, false negative rate, and false positive rate of HDC were 74.5% , 94.6% , 25.5% , and 5.4% , respectively,which were all better than those of I-FABP and DAO.There were significant differences of the incidence of SIRS ( P = 0.046) and abdominal infection (P = 0.027) among patients with strangulated intestinal obstruction, patients with simple intestinal obstruction, and patients with acute simple appendicitis, while lung infection showed no such significant difference (P = 0.728).The expression level of serum HDC was significantly higher in patients with strangulated intestinal obstruction who were also suffered from SIRS ( P = 0.000) or abdominal infection ( P =0.002) than that of uninfected patients.Meanwhile, the expression levels of serum I-FABP and DAO were significantly higher in the SIRS patients with strangulated intestinal obstruction than that of uninfected patients ( P = 0.027, P=0.017, respectively).The expression levels of HDC, I-FABP, and DAO were significantly correlated with the incideces of SIRS and abdominal infection ( all P < 0.05 ) , among which the level of HDC and the incidence of SIRS had the highest correlation (R = 0.608, P = 0.001).Conclusion HDC can be an effective indicator for diagnosing IMI in patients with intestinal obstruction.     

妊娠各期孕妇甲状腺激素变化的探讨

Objective To investigate the state of thyroid hormones and thyrotropin level in different gestation periods. Methods FT3, FT4, TSH and hCG were measured in 623 patients by ACS-180 automatic chemiluninescence analyzer. The urinary iodine was random measured by the use of iodine catalytic reaction mechanism of Arsenic-Cerium, rapid urinary iodine was detected with cold digestion method of quantitative detection. Result Serum FT3,FT4 ,and hCG levels in the first and second trimester were higher than in the third trimester ( F =53. 19,78.00,58.77, P ＜0. 05) ,and TSH levels were lower than in the third trimester ( F =5.68, P ＜0.05). The incidence of thyrotropin level disorder was 7. 54%, there were no significant differences in the gestation periods ( χ2 = 3.92, P ＞ 0. 05 ). The incidence of subclinical hypothyroidism,subclinical hyperthyroidism, hypothyroidism and hyperthyroidism were 3.53%, 2. 57%, 1.12% and 0. 32%, respectively. The incidence of subclinical hypothyroidism in the first and third trimester was high, while the incidence of subclinical hyperthyroidism in the second trimester was high. Conclusions Thyrotropin level in the third trimester was lower than that of the first and second trimester. HCG changes were not the main reason that caused the thyrotropin level disorder during the pregnancy.     

Chinese adults′ cognition of respiratory diseases in Healthy China Initiative (2019—2030)CSCD

Objective To investigate the cognition of Chinese adults on respiratory health in the Healthy China Initiative (2019—2030). Methods From January 2022 to February 2022, the electronic questionnaire was distributed by using the two‑dimensional code on the Wechat platform. The questionnaire involving the respiratory health part of the Healthy China Initiative (2019—2030) was conducted among people aged ≥18 years (except pregnant and postpartum women) in 32 provincial administrative regions in China. A total of 5 892 questionnaires were distributed, 4 754 valid questionnaires were recovered. Chi square test was used to analyze the basic situation of the survey subjects, the cognition of people in different regions to the content, the correlation between physical activity and age stratification, the correlation between smoking cessation and disease, and the common cognitive misunderstanding of chronic obstructive pulmonary disease. Results Of the 4 754 subjects in this study, 3 462 were from urban area and 1 292 were from rural area. In terms of active participation in lung cancer screening, regular physical examination, no awareness of reducing risk factors exposure, isolation of pulmonary tuberculosis patients, reduction of travel of pulmonary tuberculosis patients, wearing masks when contact with pulmonary tuberculosis patients, active learning of medical and health care knowledge, and timely medical treatment in case of health problems, the cognition status of people in the city was better than people in the rural areas (all P< 0.05). The compliance rates of moderate intensity physical activity and high intensity physical activity were 72.7% and 83.8%, respectively, among the population aged 18—64 years old; the compliance rates of moderate intensity physical activity and high intensity physical activity were 82.3% and 85.7%, respectively, among the population aged≥65 years old; the proportions of light intensity physical activity in people aged 18—64 and ≥65 years old were 65.2% and 87.6%, respectively. There was a correlation between whether the patients had respiratory diseases and whether they were troubled by respiratory diseases for a long time and their determination to quit smoking (all P<0.05); the rate of common misconceptions about chronic obstructive pulmonary disease among Chinese adults was between 13.0% and 38.0%, and the rate of misconceptions among people with college education or above who believed that even if the cognition of chronic obstructive pulmonary disease was improved and the diagnosis and treatment were standardized, the treatment of the disease was still ineffective was as high as 30.3%; in addition, the rate of two cognitive misunderstandings (when the symptoms of chronic obstructive pulmonary disease were mild, no intervention was needed; the medication can be discontinued when the symptoms were relieved) of people with college education or above were 24.2% and 25.8%, respectively, which were higher than those with primary school education and junior high school education (all P<0.05). Conclusions The cognition of Chinese adults on early screening of respiratory diseases, reduction of exposure to risk factors, prevention and control of tuberculosis, and acquisition of scientific knowledge in the Healthy China Initiative (2019—2030) is generally poor. The participation of people <65 years old in high‑intensity physical activities is insufficient, and the cognition of people with high education level does not show advantages. © 2021 Journal of Clinical Otorhinolaryngology Head and Neck Surgery. All rights reserved.     

大鼠肾细胞分离制备及培养方法的比较

目的比较胶原酶消化法、胰蛋白酶消化法和胶原酶、胰蛋白酶两步消化法(简称两步消化法)对大鼠肾细胞分离效果及细胞产量的影响,以及不同培养基(MEM、RPMI1640)、鼠龄(乳鼠、成年鼠)对细胞产量、质量、增殖率和乳酸脱氢酶(LDH)漏出量的影响。方法分别对两组Wistar大鼠(乳鼠和成年鼠)用三种不同的消化方法制备大鼠肾组织细胞悬液,分别在MEM培养液和RPMI1640培养液中原代培养,培养48小时之后第一次传代,观察细胞质量,计算细胞产量,MTT法检测传代细胞24~48小时之间的增殖率,生化分析仪检测传代细胞第1~7天培养液中LDH漏出量。结果胶原酶消化法和两步消化法的肾细胞产量显著高于胰蛋白酶消化法(P<0.01),两步消化法的细胞质量优于胶原酶消化法,MEM培养液和RPMI1640培养液对细胞产量、增殖率和LDH漏出量的影响均无显著性差异,乳鼠的肾细胞产量显著高于成年鼠(P<0.01)。结论两步消化法对于肾细胞的分离制备尤为适合,MEM培养液和RPMI1640培养液均适于肾细胞的培养,乳鼠比成年鼠更适合肾细胞的制备。     

改良的人脐带间充质干细胞培养方法

目的:建立人脐带间充质干细胞(hUCMSCs)的分离和培养方法,探讨hUCMSCs的成脂成骨分化潜能。方法:用酶消化法、传统组织块法和改良组织块法从人脐带中分离间充质干细胞,流式细胞仪检测其免疫表型;用不同的培养体系诱导hUCMSCs向成骨细胞及成脂细胞分化,并对其进行鉴定。结果:改良的hUCMSCs培养方法培养细胞纯度更高,在相同的培养时间内,改良组织块法所获得的细胞数量是酶消化法的2～3倍,是传统组织块法的20～30倍,细胞呈长梭形生长;高表达CD73、CD90、CD44、CD105,不表达CD31、CD45、CD34;茜素红染色及油红O染色证实了hUCMSCs可分化为脂肪细胞和成骨细胞。结论:建立了hUCMSCs的培养新方法,证实了hUCMSCs的增殖能力和多向分化潜能,为其治疗应用提供支持。     

大鼠体外多器官细胞共培养模型的建立

目的 建立大鼠体外多器官细胞共培养方法.方法 采用胶原酶消化法、支气管灌洗分离法、两步消化法等方法分离大鼠原代肝细胞、肾细胞、心肌细胞、肺泡巨噬细胞和真皮成纤维细胞,用锥虫蓝染色法检测细胞分离成活率,用单层贴壁法分别培养;采用飞片法,将5种细胞的飞片放入同一培养皿,用体积分数为10%的FBS DMEM培养基培养7 d,用噻唑蓝(MTT)比色法比较原代细胞在单独培养和共培养时的生长情况.结果 建立的大鼠原代肝细胞、肾细胞、心肌细胞、肺泡巨噬细胞、真皮成纤维细胞分离方法稳定,细胞分离成活率均达90%,其中胶原酶消化法培养肝细胞的存活率达90.3%,两步消化法培养肾细胞的细胞存活率达91.9%,心肌细胞的胶原酶消化法细胞存活率达93.0%.3～15 d的心肌细胞搏动频率比较,差异无统计学意义(P>0.05),差速贴壁法培养的肺泡巨噬细胞存活率可达90.8%,以胶原酶消化法培养的原代真皮细胞存活率达92.7%,细胞生长状态良好.5种原代细胞的多器官细胞共培养结果显示,共培养时细胞生长增殖情况良好,单独培养与共培养细胞生长曲线基本重合.结论 所建立的大鼠多器官细胞共培养方法是成功的.     

大型成年哺乳类动物绵羊阴道平滑肌细胞的原代培养改进方法研究

目的探索原代培养成年大型哺乳动物绵羊阴道平滑肌细胞(vaginal smooth muscle cells,VSMC)改良方法。方法成年绵羊阴道平滑肌取材,利用预消化组织块法(将组织剪碎呈1 mm3大小组织块,0.1%Ⅰ型胶原酶消化20 min,0.1%胰酶-EDTA与0.1%Ⅰ型胶原酶混合溶液再次消化30 min,将组织块种于培养瓶)原代培养VSMC,并与传统组织块法和酶消化法进行细胞萌出速度及增殖速度比较,提高VSMC原代培养效率;免疫荧光染色法和western bloting法检测平滑肌细胞标志物calponin及a-SMA表达鉴定VSMC。结果预消化组织块法获得原代VSMC速度快于传统组织块法和酶消化法,3 d可见细胞萌出,9 d可达80%细胞融合,"峰谷"特征明显,而酶消化法至12 d增殖至80%细胞汇合,传统组织块法约7 d可见细胞萌出,14 d可达80%细胞汇合;VSMC平滑肌标志物calponin和a-SMA均呈阳性表达(P0.05);VSMC可扩增代数有限,扩增7代后即表现出细胞形态改变,增殖缓慢,胞核内出现空泡,死亡细胞增加等表现。结论预消化组织块法可较快速多量获得绵羊VSMC。     

两种方法分离新生大鼠颅骨成骨细胞纯度和活性的比较

目的比较预消化组织块培养法与传统组织块培养法分离的新生大鼠颅骨成骨细胞纯度和活性的差异。方法无菌条件下取新生大鼠颅骨骨片,分传统法和预消化法进行培养。预消化法先以0.25%胰蛋白酶/0.02%EDTA预先消化,再按传统组织块培养法培养,观察细胞移出时间,采用Gomori钙钴法对原代细胞及纯化后细胞进行鉴定,对比两种方法获得的成骨细胞纯度。并测定第1、2代成骨细胞培养第10d胞浆内碱性磷酸酶活性。结果预消化法约第3d开始细胞自骨片移出,传统法约第5d开始细胞移出。两种方法分离的原代细胞碱性磷酸酶染色阳性率分别为(87.83±2.34)%以及(82.88±3.14)%,差异有统计学意义(P<0.05)。纯化后成骨细胞染色阳性率分别为(90.71±3.15)%以及(90.17±2.97)%,两组比较差异无统计学意义(P>0.05)。两种方法获得的第1、2代成骨细胞培养第10d碱性磷酸酶活性比较差异无统计学意义(P>0.05)。结论预消化组织块培养法细胞移出时间早,且所分离的原代细胞中成骨细胞纯度高。经纯化后成骨细胞纯度和活性与传统组织块培养法相似。     

子宫腺肌病病灶细胞的培养与鉴定

目的原代培养及鉴定子宫腺肌病病灶细胞,为研究子宫腺肌病发病机制与药效学提供新的理想实验模型。方法采用胶原酶消化法对人子宫腺肌病细胞进行原代培养,并采用免疫细胞化学技术鉴定。结果成功培养了6例子宫腺肌病细胞并均经免疫细胞化学法鉴定证实,成功率为75％;第1代与第8代细胞的生长曲线基本一致。结论胶原酶消化法培养的子宫腺肌病细胞性能稳定,后续研究可选择3～6代细胞作为药物干预模型,细胞传代后4—5天加药物干预,选择24小时作为药效学作用的检测时间点。     

20例人早孕绒毛滋养细胞的体外培养

目的建立快速、有效的绒毛滋养细胞体外培养的方法。方法采用0．25％胰酶和Ⅱ型胶原酶混合消化法消化绒毛组织,用胰蛋白酶消化传代体外培养,通过光镜对培养出的细胞进行形态评价;用细胞角蛋白和波形蛋白单克隆抗体检测细胞纯度。结果联合消化法的细胞生长迅速,接种6～8d可以传代,原代细胞生长时间明显缩短,所得细胞纯度达95％。结论联合消化法是一种较有效的人早孕绒毛滋养细胞培养方法。     

胎盘Hofbauer细胞的体外培养和鉴定

目的 通过对早孕胎盘绒毛膜Hofbauer细胞的分离,纯化和培养,寻找一种稳定、简便的,可获得较高纯度Hofbauer细胞的培养方法,以满足后续实验要求.方法 通过胰酶/胶原酶联合消化法对妊娠6～10周绒毛组织进行消化,获得单细胞悬液,根据不同细胞贴壁时间的差异,对Hofbauer细胞进行纯化,并通过免疫组织化学法对Hofbauer细胞进行鉴定.结果 获得了较高纯度的胎盘Hofbauer细胞,免疫组化法鉴定CD68阳性,细胞生长状态良好.结论 利用胰酶/胶原酶联合消化法及差异贴壁法可以获得满意的人胎盘Hofbauer细胞.     

镉致成骨细胞增殖、分化及矿化损伤的预后效应

[目的]探讨镉染毒和染毒中止后成骨细胞增殖、分化及矿化等生物学功能的改变。[方法]采用混合酶消化法,分离sD大鼠颅盖骨成骨细胞,在5％CO2、37℃条件下培养24h后,持续镉染毒组以不同浓度的氯化镉（0～2．000μmol／L）持续染毒72h;间断镉染毒组以同样浓度的氯化镉染毒48h后,更换成无镉培养液继续培养24h,以噻唑蓝（MTT）法测定细胞增殖能力改变,用对硝基苯磷酸二钠盐（PNPP）偶氮法检测碱性磷酸酶（ALP）活性。同时,成骨细胞培养7d后,以0．500μmol／L氯化镉持续作用3—13d,然后分别停止作用10、8、6、0d,采用茜素红S进行矿化结节染色并计算面积以观察镉暴露中止后成骨细胞矿化能力损伤的恢复情况。[结果]氯化镉可明显抑制成骨细胞增殖、ALP活性及矿化能力。间断镉染毒组在停止作用24h后,成骨细胞的增殖、分化仍然明显低于对照组（P〈0．05）,与持续作用组比较没有明显改善。同样,间断镉染毒组在停止作用不同时间后,成骨细胞矿化结节数量和面积仍然明显低于对照组（P〈O．05）,与持续作用组相比没有明显恢复。[结论]镉暴露中止一定时间后,镉对成骨细胞的影响作用仍明显存在,表现为对成骨细胞增殖、分化及矿化能力的持续抑制。     

牛乳骨桥蛋白对大鼠成骨细胞的影响

目的观察乳中骨桥蛋白(osteopontin,OPN)对体外培养大鼠成骨细胞的影响。方法采用二次酶消化法体外培养成骨细胞,MTT比色法测定骨桥蛋白对成骨细胞的增殖影响,ALP检测试剂盒测定细胞的ALP活性,RT-PCR法检测细胞中OPG/RANKL mRNA的表达。结果在细胞增殖实验中,OPN2.5μg/ml与对照组比较,成骨细胞的增殖不显著(P>0.05),5.0μg/ml组和10.0μg/ml组增殖显著(P<0.05),20μg/ml组和40μg/ml组增殖作用极显著差异(P<0.01);在成骨细胞ALP活性检测中,OPN10μg/ml组和20μg/ml组在第3、5、7和9d可提高ALP的活性(P<0.05),40μg/ml组在各个时期均能提高ALP的活性(P<0.01),2.5μg/ml组和5.0μg/ml组提高ALP的活性不显著;OPN能提高OPG/RANKL mRNA的表达。结论骨桥蛋白能够促进成骨细胞增殖、ALP活性和OPG/RANKL mRNA的表达。