Objective:To establish a disk (CD) microfluidic chip detection platform for the rapid detection of CALR-1 and CALR-2 mutations in patients with cerebral infarction, and summarize its clinical application value.Methods:Based on microfluidic technology and loop mediated isothermal amplification technology, a CD microfluidic chip detection platform for simultaneous detection of CALR-1 and CALR-2 gene mutations were established, and the sensitivity, specificity, repeatability and accuracy of the platform were verified. A total of 124 patients with cerebral infarction treated in Huashan Hospital, Shanghai Medical College, Fudan University from November 2019 to March 2021 were prospectively selected into the experimental group; and 80 healthy subjects were included in the control group. The CALR-1 and CALR-2 gene mutations in anticoagulant peripheral blood samples were detected by the CD microfluidic chip. Each chip could detect 4 samples at the same time and synchronously detect 3 indexes of each sample. The detection results could be obtained after isothermal amplification for 40 min. At the same time, sequencing method was used to verify the test results, and the consistency of the results of the two detection methods was compared.Results:Using this CD microfluidic chip platform, the synchronous amplification of 3 indexes in the sample could be completed within 40 min without the need of thermal circulation, and the whole detection process of the sample could be completed within 60 min. For samples with a high concentration of target nucleic acid, typical positive signals could be visualized after amplification for 10 min, and the test results would be available within 30 minutes after receiving the samples. The detection sensitivity of CD microfluidic chip method for CALR-1 and CALR-2 mutation load concentration was 1.0% and 0.5% respectively. Nonspecific amplification was not observed for the non-target nucleic acid samples, indicating the high specificity of this method. The coincidence rates of intra and inter batch repeatability were 100% (20/20) respectively. Two samples with CALR gene mutation were found in the cerebral infarction group, both of which were CALR-1 mutations (L367fs*46). There was no CALR-1 or CALR-2 mutation in the control group. The detection results of CD microfluidic chip method were completely consistent with the sequencing verification results (100% [204/204]).Conclusions:The CD microfluidic chip method could be used for the detection of CALR-1 and CALR-2 gene mutations in clinical samples of patients with cerebral infarction. This method has the advantages of high detection sensitivity, good detection specificity, fast detection speed and high detection flux, which is helpful to clarify the etiology of patients with cerebral infarction.

Objective To investigate relativity between the epidemiology of HPV and cervical carcinoma in Dali region,Yunnan province,through detecting the 13 high-risk human papillomavirus infection and Thinprep cytologic test in 2153 cases.Methods Real-time PCR was used to detect the 13 high-risk HPV (16,18,31,33,35,39,45,51,52,56,58,59,68) in2153 cases and 1604 cases were checked with Thinprep cytologic test.Results In 2153 samples,260 cases were infected with HPV,with the positive rate of 12.08％.The highest positive rates were ＞60 years old (18.18％),then ＞20 and ≤30 years old (14.41％);there was no significant difference in the positive rate among the various age groups (P =0.384).There were 1465 negative for intraepithelial lesion ormalignancy (NILM) cases (91.33％),86 atypical squamous cells of undetermined significance (ASC-US) and atypical squamous cells cannot exclude HSIL (ASC-H) cases (5.36％),32 low-grade squamous intraepithelial lesion cases (LSIL) cases (2.00％),21 high-grade squamous intraepithelial lesion cases (1.31％) through Thinprep cytologic test.The correlation coefficient is 0.893.Conclusions The infection rate of HPV in Dali region,Yunnan Province,has no significant difference among the various age groups.Application of 13 high-risk HPV infection test combined with Thinprep cytologic test could be more effective in screening cervical carcinoma.

Objective To investigate if immunological factors associated with the false HIV screening test results in RA.Methods Subjects who attended the Rheumatology Outpatient Clinic -Internal Medicine Unit of Guanghua Integrative Hospital , from October 2013 to October 2014, who met the American College of Rheumatology /European League Against Rheumatism Criteria for RA were recruited for the study . 100 subjects with RA were recruited.Each patient underwent clinical examination and blood sampling for assessment of serum HIV screening test and Rheumatoid factors ( RF-IgA, -IgG, -IgM) and anti-cyclic citrullinated protein antibodies (anti-CCP) were purified from the plasma and detected by ELISA , Samples were collected and processed using standard protocols and were stored in the same freezer before analysis . RA patients were divided into two groups based on the titters of RF and anti -CCP:RF <18 U/ml/anti-CCP <25 U/ml group and RF >300 U/ml /anti-CCP >500 U/ml group.HIV screening tests were determined by three methods: ELISA、Immuno-colloidal Golden Method and ECLIA.The positive results were confirmed by the Changning Centers for Disease Control , Shanghai through western -blotting test.Results 100 samples detected by ELISA and Immuno-colloidal Golden Method were given negative results , 16 positive results existed in ECLIA group.There were1,12,3 positive cases in RF-IgM <18 U/ml, RF-IgM and RF-IgG >300 U/ml group(2.7%,32.4%,13.6%;P <0.01).In anti-CCP <25 RU/ml and >500 RU/ml groups there were 2 and 4 positive results(4.7%,24.6%;P <0.01).Conclusions Different HIV screening test methods would give different results , according to operation requirement using second method to determine the HIV screening result.HIV False-positivity was associated with the titers of anti -CCP and RF in RA.

Objective To investigate the percentages of T follicuLar helper cells (Tfh)and interleukin-21(IL-21)in the plasma of patients with rheumatoid arthritis,and the immunological mechanism of Tfh cells in the development of rheumatoid arthritis. Methods According to ACR and DAS28,patients with rheumatoid arthritis were divided into low,moderate and high activity group.The percentages of CD3 + CD4 + CXCR+ 、CD3 + CD4 + ICOS+ and CD3 + CD4 + CXCR+ ICOS+ Tfh cells were detected by Flow Cytometry.While the levels of IL-21 、RF-IgM and anti-CCP in plasma were measured by ELlSA test.The analysis was performed by t-test and Spearman′s correlation analysis.Results The expression of CD3 + CD4 + CXCR+ ICOS+ Tfh cells in PBMCs of rheu-matoid arthritis was significantly higher than that in the normal controls(P <0.05).Meanwhile the results of the three rheumatoid arthritis groups(low,moderate and high activity groups)showed that the expression of Tfh increased accordingly(P <0.05).The expression of Tfh in rheumatoid arthritis was positively related with the levels of IL-21 ,ESR,CRP,RF and anti-CCP respectively. Conclusion The expression of Tfh and IL-21 increases significantly and is closely related to the disease activity in rheumatoid ar-thritis.The results indicate that the abnormality of Tfh may play an important role in the pathogenesis of rheumatoid arthritis.

In order to evaluate the biological activity in vitro and the antitumor effects of 131I-conditionally replicating oncolytic adenovirus KH901 on HepG2 human hepatoma xenografts, the leves of GM-CSF expression were determined by ELISA method. A panel of tumor and normal cells was infected with recombinant adenovirus KH901 at MOI of 10 PPC. The medium was harvested to determine the bioactivity of GM-CSF after 24 hours. Nude mice bearing HepG2 human hepatoma xenografts were given 131-KH901. Antitumor effects were assessed using endpoints of tumor growth delay. The data showed that after 24 hours 131-KH901 replicated hugely in tumor cells and produced significant amount of GM-CSF 183.27 +/- 6.90 pg/ml, while producing very small amount of GM-CSF 20.44 +/- 0.77 pg/ml in normal cells. In the treatment of tumor, 131I-KH901 showed higher restraint rate (71.3%) compared to 131I (22.7%) or KH901 (52.7%). Therefore, 131-KH901 can inhibit the growth of human hepatoma cell in nude mice and it may be a potential drug for treating liver cancer.
Adenoviridae ; genetics ; metabolism ; Animals ; Female ; Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; metabolism ; Hep G2 Cells ; Humans ; Iodine Radioisotopes ; Liver Neoplasms, Experimental ; diagnostic imaging ; pathology ; virology ; Male ; Mice ; Mice, Nude ; Oncolytic Virotherapy ; Oncolytic Viruses ; genetics ; metabolism ; Radionuclide Imaging

Objective To study the apoptosis mediated by cisplatin on human colorectal tumors (HCT116) cell line and its mechanisms in vitro. Methods The apoptosis levels of HCT116 cells mediated by cisplatin at vari-ous time and in different concentration were measured by M30-ApoptosisTM -ELISA-kits and flow cytometry assay. The expressions of the protein p53,p21 and Bcl-2 were assessed through Western-Blotting. Results Cisplatin in-hibited the proliferation in a time-and dose -dependant manner ( F = 1129. 383, P = 0. 000 and F = 125. 267, P = 0. 000, respectively). The sub-G1 peak detected by flow cytometry at 24,48,72 hours of the apoptosis rates showed a significant difference between the experimental group and the control group (χ2= 5. 669,14.110,12. 221, P = 0. 010,0.003,0. 000,respectively). We found significant differences on the HCT116 cell growth between different time under cisplatin effect (χ2 = 14.008 ,P =0. 003 ). There were significant difference on the CK18-Asp237-Asp396 re-leased by HCT116 cell between different casplatin concentration (F =48. 667 ,P =0.000) as well as different times ( F = 1194. 394, P = 0.000). The Western-Blotting results indicated that the expression level of the p53 ( t = 9.873, -2.906,7. 229,2.776,P =0.000,0. 007,0. 000,0. 011 ) and p21 (t = - 10. 692, - 8. 867, - 15. 063, - 16.281, P = 0. 000,0.001,0.000,0.000 ,respectively) increased gradually, while there are no effect on the expression of the protein Bcl-2(t=1.429,2.011,2.247,2.001,P=0. 178,0.069,0.053,0.062,respectively). Conclusions Cis-platin can induce the apoptosis on HCT116 cells and thus inhibit the reproduction of tumor cells by recovering the function of p.53.

In this research was developed high efficiency method using 125I for directly labeling KH901, a tumor-specific oncolytic recombinant adenovirus, biodistribution of 125I-labeled compound in normal mice was investigated. 125I-KH901 was prepared by N-bromosuccinimide labeling method to find the optimal ratio of labeling response. The compounds were isolated and purified by Sephadex-G10 agarose and the radiochemical purity of compounds was analyzed by paper chromatography. The radioactivity biodistribution in mice was measured at different times after caudal vein injection with 0.1ml 125I-KH901. The labeling yield of 125I-KH901 was 78% and the radiochemical purity was 95% after purification by Sephadex-G10 agarose. Biodistribution revealed that the uptake of 125I-KH901 in liver was higher than in other organs at all time points of the experiment. 125I-KH901 was mainly concentrated in liver, kidneys, spleen and lung. It can be seen that N-bromosuccinimide labeling method is an optimal method with simple steps and high labeling yield in labeling KH901 with 125I. 125I-KH901 has a biodistribution trait which is an advantage to treating liver tumors.
Adenoviridae ; genetics ; physiology ; Animals ; DNA, Recombinant ; genetics ; Female ; Genetic Vectors ; genetics ; Iodine Radioisotopes ; pharmacokinetics ; Male ; Mice ; Mice, Inbred BALB C ; Oncolytic Viruses ; genetics ; physiology

This study was undertaken to explore and compare the radiochemical behavior and biological property of antisense oligonucleotide (ASON) labeled with Technetium-99m using two methods: N-hydroxysuccinimidyl S-acetylmercaptoacetyltriglycline (NHS-MAG3) versus hydrazino nicotinamide derivative (SHNH). After SHNH and NHS-MAG3 were synthesized, ASON was labeled with Technetium-99m using SHNH and NHS-MAG3 as a bifunctional chelator, separately. The stability in vivo and in vitro, the combination with plasma albumen of rabbit, the biodistribution in BALB/ C mice and the HT29 cellular uptake were compared between labeled compound 99mTc-SHNH-ASON, using SHNH as a bifunctional complex reagent, and 99mTc-MAG3-ASON, using NHS-MAG3 as a bifunctional chelator. The results revealed that the labeling rate and the stability of 99mTc-MAG3-ASON were evidently higher than that of 99mTc-SHNH-ASON (P < 0.05), the combination rate of 99mTc-MAG3-ASON with plasma albumen was markedly lower than that of 99mTc-SHNH-ASON (P < 0.05); the biodistribution of 99mTc-MAG3-ASON was markedly lower than that of 99mTc-SHNH-ASON in blood, heart, stomach and intestines (P < 0.05), slightly lower than that of 99mTc-SHNH-ASON in liver and spleen (P > 0.05), and markedly higher than that of 99mTc-SHNH-ASON in kidney (P < 0.05); the HT29 cellular uptake rates of 99mTc-MAG3-ASON was markedly higher than that of 99mTc-SHNH-ASON (P < 0.05). Therefore, the radiochemical behavior and biological property of 99mTc-MAG3-ASON labeled using NHS-MAG3 is better than that of 99mTc-SHNH-ASON labeled using SHNH.
Animals ; Colonic Neoplasms ; metabolism ; pathology ; Glycine ; analogs & derivatives ; chemistry ; pharmacokinetics ; Humans ; Isotope Labeling ; methods ; Mice ; Mice, Inbred BALB C ; Niacinamide ; analogs & derivatives ; chemistry ; pharmacokinetics ; Oligonucleotides, Antisense ; chemistry ; pharmacokinetics ; Radiopharmaceuticals ; chemical synthesis ; pharmacokinetics ; Succinimides ; chemistry ; pharmacokinetics ; Technetium Tc 99m Mertiatide ; chemistry ; pharmacokinetics ; Tumor Cells, Cultured

This review aims to evaluate the quality of studies assessing the value of 99mTc-MIBI myocardial perfusion imaging in the diagnosis of coronary artery disease. OVID (1956 to 2006), CBMdisc (1978 to 2006), CNKI (2005 to 2006) and VIP (2005 to 2006) for relevant studies in English and Chinese were searched and identified. Quality assessment of diagnostic accuracy studies (QUADAS) items were used. Studies were classified and Meta-disc software was used to analyze sensitivity, specificity, positive likelihood ratio and negative likelihood ratio for the pooled analysis and heterogeneity test, then Asymmetric SROC curves were drawn for those without heterogeneity. In 29 articles included, the results of the pooled analysis showed that, as for rest, exercise and drug myocardial perfusion imaging, the pooled LR + were 2.209, 4.334 and 5.508, the pooled LR- were 0.224, 0.141 and 0.195, and for dipyridamole myocardial perfusion imaging, the pooled LR+ and LR- were 5.031 and 0.193, respectively. Besides, for stress myocardial perfusion imaging among the patients without myocardial infarction history, the pooled LR+ and LR- were 6.176 and 0.199, respectively. The biases from the 29 studies were mainly due to diagnostic test results review bias; variations were probable and were correlated with the spectrum of disease and inclusion criteria; the quality of report was moderate. The conclusion is that 99mTc-MIBI stress MPI, especially dipyridamole MPI, is valuable for diagnosing coronary artery disease.
Coronary Artery Disease ; diagnostic imaging ; Female ; Humans ; Male ; Myocardial Perfusion Imaging ; methods ; Radiopharmaceuticals ; Technetium Tc 99m Sestamibi

For a long time past viruses have been recognized as being tumoricidal. At present, researchers are still pursuing studies and constructing more suitable oncolytic viruses for treating different malignant tumors. Herpes simplex virus type 1 (HSV-1) has been known as the most potential oncolytic virus among all the viruses. In this overview, we summarize the current situation of oncolytic viruses, the biology of HSV-1, its construction and application of its recombinant, and we debate on the feasibility and prospect of HSV-1 mutants labeled with radionuclides for cancer therapy.
Herpesvirus 1, Human ; genetics ; physiology ; Humans ; Mutation ; Neoplasms ; radiotherapy ; Oncolytic Virotherapy ; methods ; trends ; Oncolytic Viruses ; genetics