AIM: To investigate the preventive and therapeutic effects of Hawthorn flavone on hyperlipidemia and atherosclerosis rats. METHODS: The atherosclerosis model was established by high fat diet plus vitamin D2. The blood lipid levels, heart index, atherosclerosis index (AI1, AI2) and coronary heart index were measured in each group. The histopathological changes of aorta were observed by oil red O staining, HE staining and Masson staining. ELISA experiments were used to detect IL-6, ICAM-1, MCP-1 and VCAM-1 protein level. RESULTS: Compared with normal group, total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL-C), heart index, atherosclerosis index (AI1, AI2) and coronary index in atherosclerosis model group were significantly increased (P<0.01), while high density lipoprotein cholesterol (HDL-C) was significantly decreased (P<0.01). The pathological score of aorta and the degree of fibrosis were significantly increased (P<0.01). Compared with model group, TC, TG, LDL-C, heart index, atherosclerosis index (AI1, AI2) and coronary heart index were significantly decreased (P<0.01), and high-density lipoprotein cholesterol (HDL-C) was significantly increased (P<0.01) in medium, high dose hawthorn flavonoids and atorvastatin groups. The pathological score of aorta significantly decreased and the degree of fibrosis significantly improved (P<0.01). The variation trend of blood lipid levels in hyperlipidemia rats is basically consistent with atherosclerotic rats. Meanwhile, compared with model group, the medium, high dose hawthorn flavonoids and atorvastatin groups could significantly inhibit the expression levels of IL-6, MCP-1, ICAM-1 and VCAM-1 adhesion molecules (P<0.01). CONCLUSION: The hawthorn flavone can inhibit the formation of aortic endothelial atherosclerotic plaque, reduce the degree of fibrosis and inflammation of atherosclerotic plaque in rats, and achieve the purpose of anti-atherosclerosis. Meanwhile, the hawthorn flavone has the effect of regulating blood lipid.

Hepatocellular carcinoma is a common malignant disease in clinical practice, and portal vein tumor thrombosis (PVTT) is one of the important factors affecting the prognosis of hepatocellular carcinoma. PVTT has strong oncologic characteristics and is highly susceptible to extrahepatic metastasis, complicating portal hypertension, leading to gastrointestinal bleeding or liver failure and causing death. In this paper, we review the formation mechanism of hepatocellular carcinoma combined with PVTT in terms of local anatomy, hemodynamics, molecular biology and tumor microenvironment to provide effective reference for clinical treatment.

目的：通过 CRISPR/Cas9 技术构建前列腺癌 PC3 细胞 TFDP3 基因敲除的稳转株，探讨抑制 TFDP3 表达对 PC3 细 胞周期、凋亡、迁移和侵袭能力的影响。方法：通过生物信息学筛选 sgRNA，通过 CRISPR/Cas9 技术、构建抑制 TFDP3 基因表达 的 sgRNA-Cas9 共转染慢病毒，感染 PC3 细胞后筛选获取稳转细胞株。通过流式细胞术对 TFDP3 基因敲除的实验组与空白对照 组进行细胞周期和凋亡检测，并进一步通过划痕实验和 Transwell 实验进行细胞迁移和侵袭能力检测。结果：通过生物信息学 筛选获得 3 条 sgRNA，其中 sgRNA2 有明显的抑制前列腺癌细胞基因表达的功能；通过 CRISPR/Cas9 技术成功构建了基于 CRISPR/Cas9 介导的 TFDP3 低表达的 PC3 细胞稳转株。抑制 TFDP3 基因表达后，相比于对照组，KO 组中 G0/G1 期细胞 百分比增加、G2/M 期细胞百分比下降（P<0.05 或 P<0.01），细胞凋亡率显著升高（P<0.05），细胞迁移率明显下降 [24 h 迁移率： (44.00±1.60)% vs (65.00±4.40)%，P<0.01]，穿过聚碳酸酯膜的侵袭细胞数明显下降 [（185.89±11.71）vs （248.33±11.95）个， P<0.01]。结论：通过 CRISPR/Cas9 技术抑制 TFDP3 基因表达后，PC3 细胞发生周期阻滞、凋亡率也有所增加、迁移和侵袭能力 显著减弱，提示 TFDP3 是一个前列腺癌促癌基因。

OBJECTIVE: To analyze the clinical phenotype and genetic basis of a consanguineous pedigree affected with hereditary coagulation factor XII (FXII) deficiency. METHODS: Following extraction of genomic DNA, all exons and flanking regions of F12 gene were subjected to PCR amplification and Sanger sequencing. ClustalX-2.1-win and MutationTaster software was used to analyze the conservation and impact of the variants on protein function. RESULTS: DNA sequencing showed that the proband carried a homozygous g.6753-6755delACA deletion (p.252delAsn) in exon 9 of the F12 gene, for which her father, mother and brother were heterozygous carriers. The same deletion was not found in her sister. CONCLUSION The homozygous p.252delAsn deletion probably underlies the hereditary FXII deficiency in this pedigree.

Objective: To optimize the existing methods of isolation and purification for exosomes from urine and explore the effects of different storage conditions on the content of exosomal RNA in urine. Methods: The exosomes in human urine samples were extracted by different precipitation method, i.e., precipitation following first concentrating and direct precipitation, respectively, and the separation efficiency and cost of the two methods were compared. ExoQuick-TCTM precipitation kit was used to extract exosomes. Nanoparticle tracking analysis technique (NTA) was used to detect the concentration and particle size distribution of exosome. Dynamic light scattering (DLS) was used to detect the potential of exosome. Transmission electron microscopy (TEM) was used to observe morphology of exosomes. western blot was used to analyze the exosomal marker molecules CD63 and Alix. The extraction method of the precipitation following first concentrating was used to verify the reliability of the optimized method in 10 clinical urine samples . Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression levels of exosomal RNA marker let-7c and PSA mRNA in the urinary exosomes from 20 patients with prostate cancer after repeated freeze-thaw (0 [i.e., fresh], 1 , 3 and 5 times) and 9 patients with prostate cancer frozen at -80 ℃ for different time (0 [i.e., fresh], 1, 2 and 4 weeks), and were statistically analyzed by Wilcoxon rank sum test for differences between the 2 groups. Results: The size distribution of exosomes extracted by the two methods was 30 to 150 nm by NTA, both of which were displayed as single peaks. The results of DLS showed that the potentials of exosome extracted by the two methods were negative values. The size of the exosomes extracted by the two methods was consistent observed under TEM namely the diameter distribution was 30 to 150 nm. western blot analysis confirmed that CD63 and Alix, the exosome labeling molecules, existed in the optimized method. The concentration of exosomes extracted from the 10 urine samples all reached 10 9 to 10 11 particles/mL. The contents of let-7c and PSA mRNA in exosomes decreased significantly after 5 freeze-thaw cycles, and the Z values were -1.79 and -1.73, respectively (P＜0.05). The RNA content of the exosomes remained stable after freezing at -80 ℃ for 1 month. Conclusion The optimized exosome extraction method could reduce greatly the cost under the premises of ensuring the concentration and quality of exosomes. The isolated exosomes may keep stable RNA content after freezing at -80 ℃ for a short time, but could not be frozen and thawed repeatedly for more than 5 times.

Objective To evaluate the diagnostic value of circulating tumor cells(CTCs) in prostate cancer (Pca) through studying the relationship between CTCs and Gleason scores and pathological TNM stage in Pca patients. Methods A total of 238 patients including 161 Pca patients as cancer group, 35 male patients with benign prostatic diseases as benign group and 42 male with non-prostate disease as control group, who were treated in our hospital from July 2016 to January 2018,were enrolled. Venous blood of every patient was collected and CTCs were enriched and identified by immunocytochemistry CD45 capturing leukocyte and fluorescence in situ hybridization with chromosome 8 (CEP8-FISH). Cells displaying CD45-/DAPI+/CEP8>2 were characterized as CTCs. One-way ANOVA was used to exam the correlations of the number of CTCs with Gleason scores and pathological TNM stage. Results CTCs ≥2 were detected in 74.53％(120/161) of Pca patients and 20.00％(7/35)of benign prostatic diseases patients and 7.14％(3/42)of control group (χ2=79.605,P<0.05). In group Gleason scores 6, the numbers of CTCs were 2.00 ± 2.42, the ratios of CTCs≥5 and tetraploid were 13.33％ (2/15)and 26.67％(4/15) respectively. In 7 scores group, the results were 3.14±2.68,17.72％(14/79) and 34.18％(27/79)respectively;In 8 scores group, the results were 3.57 ± 2.70, 33.33％(7/21)and 42.86％ (9/21)respectively; In 9 scores group, these three results were 4.65±4.41, 43.48％(20/46) and 45.65％(21/46)respectively. The numbers of CTCs in the≤pT2b (20), pT2c(27), pT3a(19), pT3b(16)and≥pT4(12) groups were 2.25±2.45, 3.56±2.79, 4.05±3.47, 4.69±2.12 and 5.17±3.21 respectively. The ratios of CTCs≥5 were 25.00％(5/20), 25.93％(7/27), 26.32％(5/19), 50.00％(8/16) and 58.33％ (7/12)respectively. The proportions of tetraploid were 20.00％(4/20), 25.93％ (7/27), 31.58％(6/19), 50.00％(8/16) and 58.33％(7/12) respectively. There were significant differences between CTC and Gleason scores (F=3.200, P<0.05)and pathological stage (F=2.673, P<0.05). The ratios of CTCs≥5 increased with the increase of Gleason scores (χ2=11.592, P<0.05). Conclusions The detection of CTCs could be used for the differential diagnosis of Pca and benign prostatic disease. There were notable correlations between the numbers of CTCs and Gleason scores and pathological stage in Pca patients, especially between CTCs≥5 and Gleason scores.

Objective To analyze the expression of urine exosomal miR-375 in prostate tumors and investigate its clinical utility.Methods A total of 45 patients with PCa,24 with benign prostate hyperplasia (BPH) and 24 healthy individuals were enrolled into this study.Exosomes were isolated from the urine of PCa,BPH and healthy individuals and the total RNA was extracted from the exosomes.The exosomal miR-375 expression was assessed by quantitative real-time PCR and analyzed with the comparative quantification cycle method (2-△△CT).We performed comprehensive biostatistical analyses to explore the clinical value of miR-375 in prostate cancer.Results The urine exosomal miR-375 expression was significantly downregulated in the patients with PCa compared with BPH and the healthy controls (P ＜ 0.01).No statistically significant difference of the urine exosomal miR-375 expression levels between the patients with BPH and healthy individuals was observed (P ＞ 0.05).The urine exosomal miR-375 expression level was also found to be associated with clinical stage and bone metastasis status of the patients with PCa (P ＜0.05),and with the increase of Clinical stage.The expression level of miR-375 decreased.No significant relationship was detected between miR-375 level and the patient's age,gleason score and serum prostate-specific antigen level (P ＞ 0.05).Receiver operator characteristic analyses demonstrated that the urine exosomal miR-375 expression could better differentiate PCa from BPH patients:AUC 0.715 (95％ CI:0.589-0.842) vs PSA AUC 0.632 (95％ CI:0.492-0.771) (P＜0.01).Conclusion The urine exosomal miR-375 could serve as a non-invasive biomarker for the diagnosis of PCa.

Objective To investigate the correlation between the results of HBsAg positive results among different detection systems,and provide reference for the analysis of clinical test results and the publication of the report.Methods The HBsAg positive serum specimens with the quantitation result lower than 80 COI were detected by Roche Cobas e602 electrochemical luminescence analyzer.All the specimens were also detected by Roche Modular e170 electrochemical luminescence analyzer.The differences of detecting results were compared and performed the linear correlation analysis.Results The experimental results of two detection systems are R2 =0.933.The positive coincidences of Group A,B,C,D and E were 60.60％,92.72％,96.66％,96.66％ and 100％,respectively.The positive coincidence of the males was 87.66％,while the positive coincidence of the females was 70.96 ％.The positive coincidence of the males was significantly higher than the females (P＜0.05).Conclusion The HBsAg detecting results of Roche Cobas e602 and Roche Modular e170 electrochemical luminescence analyzer had high correlation.The results higher than 10 COI had 100％ positive coincidence rate,however the results between 1 and 10 COI were not.The result between 1 and 10 COI may lead to controversial results,suggestions for further checks.

Objective To investigate the changes of peripheral blood tacrolimus concentration and lymphocyte subsets in the uterus transplant recipient,and provide the evidence for monitoring the immune status after uterus transplantation.Methods The peripheral blood tacrolimus concentrations of the uterus transplant recipient during 1 year after transplantation were measured with the microparticle enzyme immunoassay (MEIA).Meanwhile,the whole blood cell counts and lymphocyte subsets were determined by the blood analyzer and flow cytometer,respectively.Results The blood tacrolimus concentrations of the uterus transplant recipient in the first month and second month after transplantation were (13.51 ± 3.92) ng/mL and (15.58 ± 1.19) ng/mL,respectively.The lymphocyte absolute counts were normal before transplantation.At the fifth day after transplantation,the counts of CD3 + T lymphocytes,CD4 + T lymphocytes,CD8 + T lymphocytes and NK cells and the ratio of CD4/CD8 were significantly decreased.One week after transplantation,the counts of CD4 + T lymphocytes were recovered to the normal range and maintained,but its recovery was slower than that of CD8 + T lymphocytes.The ratio of CD4/CD8 ranged from 0.4 to 0.8 during 10 days after transplantation,and increased and maintained between 0.8 and 1.1 after that.The counts of NK cells increased gradually from the 10th day after transplantation,but still did not recover to the level before transplantation even at the 20th day after transplantation.However,the counts and percentages of B lymphocytes did not decrease but increased at the fifth day after transplantation,and recovered to normal gradually from the 10th day after transplantation.There was no significant correlation between the CD3 + T lymphocyte count and blood tacrolimus concentration.Conclusion The dynamic changes of blood lymphocyte subsets and tacrolimus concentration exist in the uterus transplant recipient,which need to be further verified by a large amount of clinical data.

Objective To investigate the correlation of HCV-RNA with detection indexes HCV-Ab and HCV-cAg in its clini-cal application effect among patients with hepatitis C.Methods HCV-cAg and HCV-Ab in 140 cases of HCV-RNA were detected by enzyme linked immunosorbent assay in cases of PCR,which were detected by real-time fluorescence quantitative PCR.Results 127 cases in 140 cases of HCV-RNA positive serum were HCV-cAg positive,in line with the rate of 90.71%,and the cases of 110 HCV-Ab positive,in line with the rate of 78.57%.The positive detection rate of HCV-cAg with different HCV-RNA concentration was increased with the increase of HCV virus content,and the serum of different HCV-RNA concentration had no significant changes in HCV-Ab detection results.Conclusion The detection results of HCV-cAg had a high coincidence rate with HCV-RNA.Therefore detection of HCV-cAg can be as a complementary detec-tion of HCV-Ab,as the window period of HCV infection and infection in immunocompromised persons screening provides a simple,inexpensive method.At the same time it provides rapid screening for HCV infection provide diagnostic basis for those basic medical units who do not have the conditions for detection of HCV-RNA.