FSGS患儿肾组织免疫荧光分类以及沉积与疾病严重程度和预后的相关性分析

目的:研究局灶节段性肾小球硬化(FSGS)患儿肾组织免疫荧光分类以及沉积与疾病严重程度和预后的相关性。方法:本研究采用前瞻性研究,以我院2020年01月—2022年12月诊断并治疗的局灶节段性肾小球硬化患者128例作为研究对象,根据慢性肾脏病(CKD)分期标准,其中Ⅰ期患者12例,Ⅱ期患者33例,Ⅲ期患者41例,Ⅳ期患者27例,Ⅴ期患者15例,分别对不同严重程度以及不同预后患者的肾组织免疫荧光分类以及沉积情况进行比较。结果:肾功能Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ期患者的肾组织荧光分类情况之间的差异存在统计学意义(P<0.05);肾功能Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ期患者的肾组织荧光沉积情况之间的差异存在统计学意义(P<0.05);完全缓解组、部分缓解组以及无缓解组患者的肾组织荧光分类情况之间的差异存在统计学意义(P<0.05);完全缓解组、部分缓解组以及无缓解组患者的肾组织荧光沉积情况之间的差异存在统计学意义(P<0.05)。结论:FSGS患儿肾组织免疫荧光分类以及沉积与疾病严重程度和预后呈现显著的相关性,可以在一定的地区作为临床治疗方案制定的重要依据。

ELISA法和间接免疫荧光法诊断自身免疫疾病的临床对照

目的:对ELISA法和间接免疫荧法诊断自身免疫疾病的效果进行分析。方法:将2016年10月至2017年10月我院收治的40例患有自身免疫疾病患者作为此次研究对象,在入组后利用随机分组的方式将其分为A组与B组,A组患者采取ELISA法对抗核抗体进行检测,B组患者采取间接免疫荧光法对抗核抗体进行检测,并对检测结果进行分析。结果:A组患者检出抗核抗体为(2.58±0.57);B组患者检出抗核抗体为(0.65±0.65)。组间比较差异显著(P<0.05)。结论:ELISA法在诊断自身免疫病中有着重要的应用价值,有效的提高了对抗核抗体检测的林敏度及特异度,值得推广。

全自动血液培养仪联合全自动免疫荧分析仪检测在血液感染患者中的临床价值

目的:探讨对于血液感染者应用全自动血液培养仪联合全自动免疫荧分析仪进行诊断的价值。方法:选择2018年2月~2019年2月本院血液检验科收检的1100份血液样本作为研究对象,采用全自动血液培养仪进行血培养处理,并采用全自动免疫荧光分析仪检测血清降钙素原水平。分析检测结果。结果:血培养检测1100份样本,阳性样本130份,接着使用PCT检测130份阳性样本,阳性样本92份。经血培养检测的阴性样本970份,使用PCT检测970份阴性标本,阴性样本598份。从阳性样本检测出的致病菌中,占比最大的是革兰氏阴性菌,其中主要为大肠埃希菌与肺炎克雷伯菌。结论:对于血液感染患者,选择全自动血液培养仪联合全自动免疫荧分析仪进行检测,使确诊率得到很大提升,给后续治疗提供有效指导。

免疫荧光法测定反复发作性尿路感染患者外周血T淋巴细胞亚群的结果分析

目的:探讨免疫荧光法测定反复发作性尿路感染患者外周血T淋巴细胞亚群的价值。方法:研究选择84例反复发作性尿路感染患者及84例初次尿路感染患者及84例健康体检者作为观察对象,均为女性,于2017年9月~2019年9月收治,行免疫荧光法测定外周血T淋巴细胞亚群,组间比较检测结果。结果:与健康组相比,反复发作组患者CD3^(+)、CD4^(+)、CD4^(+)/CD8^(+)明显降低,CD8^(+)明显升高,而初次感染组患者CD3^(+)、CD4^(+)、CD8^(+)均明显升高,比较差异显著,P<0.05;健康组与初次感染组CD4^(+)/CD8^(+)比较无统计学意义,P>0.05。结论:反复发作性尿路感染患者外周血T淋巴细胞分布存在异常,可作为判断的可靠指标,采用免疫荧光法检测具有较高价值,为治疗法案调整提供可靠依据。

湖南省家犬感染狂犬病病毒状况调查研究

为了解湖南省家犬狂犬病病毒(RV)的感染状况,为防制狂犬病提供科学依据。本研究于2005年11月～2008年3月选择在全省18个县(市)收集市售家犬的脑组织标本,采用直接免疫荧光法(DFA)初筛检测RV抗原和套式RT-PCR方法检测RV特异性核酸进行确证。共检测家犬脑1613份,结果表明家犬RV感染率为4.15%。其中,雄性、雌性家犬RV感染率为4.68%和2.85%(p>0.05);大型、中型家犬RV感染率为4.57%和3.02%(p>0.05);成年犬、幼犬RV感染率为4.95%和1.57%(p<0.01);有、无狂犬疫苗免疫史的家犬RV感染率分别为1.29%、4.63%(p<0.05);春、夏、秋、冬季家犬RV感染率为9.56%、7.53%、2.31%、3.23%(p<0.01)。2005年～2006年、2007年～2008年各监测点家犬RV感染率分别与2006年、2007年人狂犬病发病率呈正相关(p<0.05)。检测结果表明湖南省家犬RV感染率较高(4.15%),存在地区、季节差异,而且家犬RV感染率与人狂犬病发病率呈正相关。

Hypertonic stimulation induces synthesis and release of glutamate in cultured rat hypothalamic astrocytes and C6 cells

Objective To investigate whether hypertonic saline （HS） can induce the synthesis and release of glutamate in cultured hypothalamic astrocytes or C6 cell line. Methods Astrocytes were isolated, cultured, purified and identified from the hypothalamus of newborn rat （1 day）. The astrocytes were randomly divided into five groups： isotonic （IS） and HS groups, astrocytes were incubated by IS and HS （320 mosM NaCl） medium, respectively, for 1, 3, 5, 10 or 15 rain; carbenoxolone （CBX） ＋IS and CBX＋HS groups, astrocytes were pre-treated with CBX （100 mmol/L） for 1 h at 37℃ in a 5% CO2 / 95% atmosphere, then removed to IS and HS medium, respectively, for 1, 3, 5, 10 or 15 min; Ca2＋＋HS group, astrocytes were pre-incubated with Ca2＋ （1 000 μmol/L） for 1 h at 37℃ in a 5% CO2 / 95% atmosphere, followed by a wash with isotonic FBS/DMEM, and then removed to hypertonic saline for 1, 3, 5, 10 or 15 min. The media of five groups were collected to analyze the medium glutamate concentration with high performance liquid chromatography. The astrocytes were fixed and double immunofluorescent stained with anti-glial fibrillary acidic protein （GFAP） and anti-glutamate. The C6 cells were divided into four groups： IS, HS, CBX＋IS and CBX＋HS groups, and used for quantitative measurement of glutamate in cells by flow cytometry （FCM）. Results （1） Anti-GFAP immunofluorescent signal revealed no significant difference among various time points in each group, or among the five groups. （2） The anti-glutamate immunofluorescent signal was increased in HS group and peaked at 5 min, and decreased and returned to the level of IS group at 15 rain （P 〈 0.01 vs the 5 min of HS group）. In CBX＋HS group, the glutamate intensity was higher than that in CBX＋IS and HS groups. （3） The medium glutamate concentration had no change after treatment with HS for 1 and 3 min, while increased markedly after treatment for 5 min to 15 min （P 〈 0.01 vs 1 min and 3 min）. On the contrary, the medium glutamate concentrations in the CBX＋HS or Ca2＋＋HS group were significant lower than that in the HS group （P 〈 0.01）. （4） FCM showed HS and CBX＋HS induced glutamate increase in C6 cells. Conclusion HS induced cultured rat hypothalamic astrocytes or C6 cells to synthesize and release glutamate; CBX could block glutamate release, but could not disrupt glutamate synthesis.

急性肾炎与肾炎型肾病综合征血清IgG变化观察

急性肾炎与肾炎型肾病综合征血清ＩｇＧ变化观察陈明明（浙江省杭州市第四人民医院儿科杭州市３１０００２）临床上一些具有肾病表现的急性肾炎，起病初时水肿较重，有大量蛋白尿和明显低蛋白血症，开始易误诊为肾病综合征［１］。而肾炎型肾病综合征在发病初期由于有血尿...

Localization of Phosphorylated Histone H3 at Mitosis and Meiosis in Wheat

One of the prominent cell cycle related modifications of histone proteins, whose function is correlated with chromosome condensation, is the phosphorylation of histone H3. Wheat (Triticum aestivum L.) mitotic and meiotic cells were analyzed with indirect immunoflurorescence labeling with an antibody recognizing histone H3 phosphorylated at Serine 10 to study the localization of phosphorylated histone H3 at mitosis and meiosis. Our results showed that, during mitotic division, the phosphoryiation of H3 started from early prophase and vanished at telophase, remaining mainly in the pericentromeric regions at metaphase and anaphase. During meiotic division, phosphorylation of H3 initiated at the transition from leptotene to zygotene and remained uniform, along the chromosomes from prophase I until telophase whereas it showed slightly stronger in the pericentromeric regions than along the chromosome arms from metaphase II until Lelophase II The different patterns of H3 phophorylation at mitosis and meiosis in wheat suggested that this evolutionarily conserved post-translational chromatin modification might be involved in more roles besides chromosome condensation.

家兔生殖道人工感染衣原体试验

捷克研究人员将鹦鹉衣原体绵羊流产株(Chlamydia psittaci PK-5082)悬浮液经两侧睾丸接种 10只家兔,剂量0.5ml/只。感染后122天剖杀,并采取睾丸、附睾、精囊、肾脏、肝脏和肺等样品,用直接免疫荧光试验和酶联免疫吸附试验(ELISA)检测。结果表明,有2只兔在感染后第63天由精液排出衣原体,其余的在感染后第77天排出。同时,在睾丸、附睾、肾、肝、

Effects of hepatitis B virus infection on human sperm chromosomes

AIM: To evaluate the level of sperm chromosome aberrations in male patients with hepatitis B, and to directly detect whether there are HBV DNA integrations in sperm chromosomes of hepatitis B patients.METHODS: Sperm chromosomes of 14 tested subjects (5healthy controls, 9 patients with HBV infection, including 1with acute hepatitis B, 2 with chronic active hepatitis B, 4with chronic persistent hepatitis B, 2 chronic HBsAg carriers with no clinical symptoms) were prepared using interspecific in vitro fertilization between zona-free golden hamster ova and human spermatozoa, and the frequencies of aberration spermatozoa were compared between subjects of HBV infection and controls. Fluorescence in situ hybridization (FISH) to sperm chromosome spreads was carried out with biotin-labeled full length HBV DNA probe to detect the specific HBV DNA sequences in the sperm chromosomes.RESULTS: The total frequency of sperm chromosome aberrations in HBV infection group (14.8%, 33/223) was significantly higher than that in the control group (4.3%,5/116). Moreover, the sperm chromosomes in HBV infection patients commonly presented stickiness, clumping, failure to staining, etc, which would affect the analysis of sperm chromosomes. Specific fluorescent signal spots for HBV DNA were seen in sperm chromosomes of one patient with chronic persistent hepatitis. In 9 (9/42) sperm chromosome complements containing fluorescent signal spots, one presented 5 obvious FISH spots, others presented 2 to 4signals. There was significant difference of fluorescence intensity among the signal spots. The distribution of signal sites among chromosomes was random.CONCLUSION: HBV infection can bring about mutagenic effects on sperm chromosomes. Integrations of viral DNA into sperm chromosomes which are multisites and nonspecific, can further increase the instability of sperm chromosomes. This study suggested that HBV infection can create extensively hereditary effects by alteration genetic constituent and/or induction chromosome aberrations, as well as the possibility of vertical transmission of HBV via the germ line to the next generation.

Detection of let-7a microRNA by real-time PCR in gastric carcinoma

AIM: To establish an accurate and rapid stem-loop reverse transcriptional real-time PCR (RT-PCR) method to quantify human let-7a miRNA in gastric cancer. METHODS: According to the sequence of let-7a miRNA,the stem-loop reverse transcriptional primer,the primers and quantitative MGB probes of real-time PCR were designed and synthesized. The dynamic range and the sensitivity of quantitative reverse transcriptional real-time PCR were determined. The levels of let-7a miRNA were examined in 32 gastric carcinoma samples by stem-loop RT-PCR method. RESULTS: The dynamic range and sensitivity of the let-7a miRNA quantification scheme were evaluated,the result showed the assay could precisely detect 10 copies of mature let-7a miRNA in as few as 0.05 ng of total RNA of gastric mucosa. The results of specificity analysis showed no fluorescence signal occurred even though 50 ng of human genomic DNA was added to the reverse transcription (RT) reaction. The expression level of let-7a miRNA in gastric tumor tissues was significantly lower compared to normal tissues in 14 samples from 32 patients. CONCLUSION: The stem-loop RT-PCR is a reliable method to detect let-7a miRNA which may play an important role in the development of gastric carcinoma.

Blocking NF-kB nuclear translocation leads to p53-related autophagy activation and cell apoptosis

AIM: To investigate the anti-tumor effects of nuclear factor-κB (NF-κB) inhibitor SN50 and related mechanisms of SGC7901 human gastric carcinoma cells. METHODS: MTT assay was used to determine the cytotoxic effects of SN50 in gastric cancer cell line SGC7901. Hoechst 33258 staining was used to detect apoptosis morphological changes after SN50 treatment. Activation of autophagy was monitored with monodansylcadaverine (MDC) staining after SN50 treatment.Immunofluorescence staining was used to detect the expression of light chain 3 (LC3). Mitochondrial membrane potential was measured using the fluorescent probe JC-1. Western blotting analysis were used to determine the expression of proteins involved in apoptosis and autophagy including p53, p53 upregulated modulator of apoptosis (PUMA), damage-regulated autophagy modulator (DRAM), LC3 and Beclin 1. We detected the effects of p53-mediated autophagy activation on the apoptosis of SGC7901 cells with the p53 inhibitor pifithrin-α. RESULTS: The viability of SGC7901 cells was inhibited after SN50 treatment. Inductions in the expression of apoptotic protein p53 and PUMA as well as autophagic protein DRAM, LC3 and Beclin 1 were detected with Western blotting analysis. SN50-treated cells exhibited punctuate microtubule-associated protein 1 LC3 in immunoreactivity and MDC-labeled vesicles increased after treatment of SN50 by MDC staining. Collapse of mitochondrial membrane potential Δψ were detected for 6 to 24 h after SN50 treatment. SN50-induced increases in PUMA, DRAM, LC3 and Beclin 1 and cell death were blocked by the p53 specific inhibitor pifithrin-α. CONCLUSION: The anti-tumor activity of NF-κB inhibitors is associated with p53-mediated activation of autophagy.

Update on Anti-Saccharomyces cerevisiae antibodies, anti-nuclear associated anti-neutrophil antibodies and antibodies to exocrine pancreas detected by indirect immunofluorescence as biomarkers in chronic inflammatory bowel diseases: Results of a multicent

AIM： Anti-Saccharomyces anti-nuclear associated cerevisiae antibodies （ASCA）, anti-neutrophil antibodies （NANA） and antibodies to exocrine pancreas （PAB）, are serological tools for discriminating Crohn＇s disease （CrD） and ulcerative colitis （UC）. Like CrD, coeliac disease （COD） is an inflammatory bowel disease （IBD） associated with （auto） antibodies. Performing a multicenter study we primarily aimed to determine the performance of ASCA, NANA and PAB tests for IBD diagnosis in children and adults, and secondarily to evaluate the prevalence of these markers in CoD. METHODS： Sera of 109 patients with CrD, 78 with UC, 45 with CoD and 50 healthy blood donors were retrospectively included. ASCA, NANA and PAB were detected by indirect immunofluorescence （IIF）. RESULTS： ASCA＋/NANA- profile displayed a positive predictive value of 94.2% for CrD. Detection of ASCA was correlated with a more severe clinical profile of CrD and treatment of the disease did not influence their serum levels. ASCA positivity was found in 37.9% of active CoD.PAB were found in 36.7% CrD and 13.3% CoD patients and were not correlated with clinical features of CrD, except with an early onset of the disease. Fifteen CrD patients were ASCA negative and PAB positive. CONCLUSION： ASCA and PAB detected by IIF are specific markers for CrD although their presence does not rule out a possible active CoD. The combination of ASCA, NANA and PAB tests improves the sensitivity of immunological markers for CrD. Repeating ASCA, NANA, and PAB testing during the course of CrD has no clinical value.

Liraglutide directly protects cardiomyocytes against reperfusion injury possibly via modulation of intracellular calcium homeostasis

Background Liraglutide is glucagon-like peptide-1 receptor agonist for treating patients with type 2 diabetes mellitus. Our previous studies have demonstrated that liraglutide protects cardiac function through improving endothelial function in patients with acute myocardial infarction undergoing percutaneous coronary intervention. The present study will investigate whether liraglntide can perform direct protective effects on cardiomyocytes against reperfusion injury. Methods In vitro experiments were performed using H9C2 cells and neonatal rat ventricular cadiomyocytes undergoing simulative hypoxia/reoxygenation （H/R） induction. Cardiomyocytes apoptosis was detected by fluorescence TUNEL. Mitochondrial membrane potential （AWm） and intracellular reactive oxygen species （ROS） was assessed by JC-1 and DHE, respectively. Fura-2/AM was used to measure intracellular Ca2＋ concentration and calcium transient. Immtmofluorescence staining was used to assess the expression level of sarcoplasmic reticulum Ca2＋-ATPase （SERCA2a）. In vivo experiments, myocardial apoptosis and expression of SERCA2a were detected by colorimetric TUNEL and by immunofluorescence staining, respectively. Results In vitro liraglutide inhibited cardiomyotes apoptosis against H/R. △mψ of cardiomyocytes was higher in liraglntide group than H/R group. H/R increased ROS production in H9C2 cells which was attenuated by liraglutide. Liraglutide significantly lowered Ca2＋ overload and improved calcium transient compared with H/R group, lmmunofluorescence staining results showed liraglutide promoted SERCA2a expression which was decreased in H/R group. In ischemia/reperfusion rat hearts, apoptosis was significantly attenuated and SERCA2a expression was increased by liraglutide compared with H/R group. Conclusions Liraglutide can directly protect cardiomyocytes against reperfusion injury which is possibly through modulation of intracellular calcium homeostasis.

Intestinal alkaline phosphatase in the colonic mucosa of children with inflammatory bowel disease

AIM： To investigate intestinal alkaline phosphatase （iAP） in the intestinal mucosa of children with inflammatory bowel disease （IBD）. METHODS： Colonic biopsy samples were taken from 15 newly diagnosed IBD patients and from 10 healthy controls. In IBD patients, specimens were obtainedboth from inflamed and non-inflamed areas. The lAP mRNA and protein expression was determined by reverse transcription-polymerase chain reaction and Western blotting analysis, respectively. Tissue localiza- tion of lAP and Toll-like receptor （TLR） 4 was investi- gated by immunofluorescent staining. RESULTS： The lAP protein level in the inflamed muco- sa of children with Crohn＇s disease （CD） and ulcerative colitis （UC） was significantly decreased when compared with controls （both P 〈 0.05）. Similarly, we found a significantly decreased level of lAP protein in the in- flamed mucosa in CD compared with non-inflamed mucosa in CD （P 〈 0.05）. In addition, the iAP protein level in inflamed colonic mucosa in patients with UC was decreased compared with non-inflamed mucosa in patients with CD （P 〈 0.05）. lAP protein levels in the non-inflamed mucosa of patients with CD were similar to controls, lAP mRNA expression in inflamed colonic mucosa of children with CD and UC was not significant- ly different from that in non-inflamed colonic mucosa with CD. Expression of lAP mRNA in patients with non- inflamed mucosa and in controls were similar. Co-local- ization of lAP with TLR4 showed intense staining with a dotted-like pattern, lAP was present in the inflamed and non-inflamed mucosa of patients with CD, UC, and in control biopsy specimens, irrespective of whether it was present in the terminal ileum or in the colon. However, the fluorescent signal of TLR4 was more pro- nounced in the colon compared with the terminal ileum in all groups studied. CONCLUSION： Lower than normal lAP protein levels in inflamed mucosa of IBD patients may indicate a role for lAP in inflammatory lesions in IBD. Based on our results, administration of exogenous lAP enzyme to pa- tients with the active form of IBD may be a therapeutic option.

Streptozotocin-induced expression of Ngn3 and Pax4 in neonatal rat pancreatic α-cells

AIM： To investigate the mechanism behind β-cell regeneration in neonatal rat pancreas treated with strep- tozotocin （STZ）. METHODS： Neonatal Sprague Dawley rats were intra- peritoneally injected with 70 mg/kg STZ. Body weight, pancreas weight and blood glucose were recorded every two days after the treatment. To identify the expression and location of transcription factors in the rat pancreas, double immunofluorescent staining was performed using antibodies to specific cell markers and transcription factors. RESULTS： Expression of Neurogenin 3 （Ngn3）, a marker for endocrine precursor cells, was observed by immunofluorescence in a few β-cells and many a-cells. The expression reached a peak 12 d after treatment. Pax4, a transcription factor that lies downstream of Ngn3 and plays an important role in β-cell differentiation, was also expressed in the α-cells of STZ-treated rats. We did not observe significant changes in Nkx6.1, which is essential for β-cell maturation in the treated rats. CONCLUSION： α-cells dedifferentiated into endocrine precursor cells and acquired the ability to dedifferentiate in the neonatal rat pancreas after STZ treatment.

Expression and location of α-fetoprotein during rat colon development

AIM： To investigate the expression of α-fetoprotein （AFP）, a cancer-associated fetal glycoprotein, and its involvement during rat colon development. METHODS： Colons from Sprague-Dawley rat fetuses, young and adult （8 wk old） animals were used in this study. Expression levels of AFP in colons of different development stage were detected by reversetranscriptase PCR （RT-PCR） and Western blotting. To identify the cell location of AFP in the developing rat colons, double-immunofluorescent staining was performed using antibodies to specific cell markers and AFP, respectively. RESULTS： The highest levels of AFP mRNA were detected in colons of rats at embryonic day 18.5 （e18.5）. Compared to e18.5 d, the AFP expression was significantly decreased during rat development （85% for e20.5, P 〈 0.05, 58% for postnatal day 0.5 （P〈0.5）, P 〈 0.05, 37% for P7, P 〈 0.05, 24% for P14, P 〈 0.05, and 11% for P21, P 〈 0.05） and undetected in adult rats. Only the 72-kDa isoform of AFP was detected by Western blotting, the expression pattern was similar to AFP mRNA and conformed to the results of mRNA expression. The AFP positive staining was identical to different distribution patterns in fetuses, young and adult animals and positive staining for both AFP and vimentin was overlapped in mesenchymal cells at each stage tested. CONCLUSION： This study has for the first time demonstrated that AFP is localized in the mesenchyme of rat colon from the embryo to the weaning stage by immunofluorescence and presents 72-kDa isoform in the developing rat colons by Western blotting. The dynamic expression of AFP in the various developmental stages of the colon indicates that AFP might be involved in many aspects of colon development.