Evaluation of the consistency and detection capability of seven domestic 2019-nCoV nucleic acid detection kits / 中华检验医学杂志

Objective:To compare the consistency and detection capability of seven 2019-nCoV nucleic acid detection kits, and provide reference for detection method selection of clinical laboratory and diagnosis of new coronavirus pneumonia.Methods:Two batches of pharyngeal swab samples were collected from tenpatients with confirmed infection of 2019-nCoV and 10 suspected patients with negative 2019-nCoV test results during January 29 to February 5, 2020 in Shenzhen Luohu People′s Hospital. Seven kinds of kits were labeled as ato g and used for nucleic acid detection respectively to evaluate the consistency of the test results of the clinical samples. A 2019-nCoV positive specimen was selected and diluted to 5-concentration gradient plates (Level-1 to 5) with RNase-free water. The positive detection rate and intra-batch repeatability of different brands of kits were compared.Results:The negative and positive coincidence rates of twenty clinical samples tested by six kinds of kits were 100%, and the positive and negative coincidence rate was 8/10 and 10/10 for the other kit, respectively. The results of intra-batch repeatability showed the CVs of viral loads tested by these seven kits were all less than 5%. In the concentration range of Level-1 to 3, the detection capability for open reading frame (ORF)1ab gene of Kit b,d and f was lower than Kit a,c,e and g, and the detection capability of kit e and g was the highest (14/15). The detection capability for N gene of Kit a (15/15) was higher than the other 5 kits. The comprehensive analysis of the detection capability for ORF1ab and N gene showedthat Kit d had the lowest detection capability (ORF1ab:40%,N:53%), and there was no significant difference in the detection capability of Kit a, b, c, e, and f.Conclusions:There was no significant difference in the accuracy and repeatability of the seven kits for positive samples with high viral loads, and the detection performance was good; but some kits had poor detection capability for weak positive samples. It is suggested that the weak positive samples should be rechecked by at least two manufacturers′ kits to ensure the accuracy of the results.

Analysis of clinical phenotype of a α2-globin gene mutation -IVS-Ⅱ-55 (T→G) / 临床检验杂志

Objective@#To identify a α-globin gene mutation-IVS-Ⅱ-55 (T→A) and analyze hematological characteristics of IVS-Ⅱ-55 (T→G) carriers. @*Methods@#The peripheral blood samples were collected from the members of five family and three sporadic IVS-Ⅱ-55(T→G) carriers for the analysis of RBC parameters and hemoglobin electrophoresis. Gap-PCR, PCR-RDB (reverse dot blot) and DNA sequencing were carried out for the identification of gene deletion and mutation of α-globin and β-globin. @*Results@#The results of RBC parameters of five infant probands which presented with microcytic hypochromic anemia were below the normal reference interval. One of the adult carriers of IVS-Ⅱ-55 (T→G) heterozygote alone presented with microcytic hypochromic anemia, and the others showed normal RBC parameters. The hematological phenotype index (MCV, MCH and HbA 2 ) of one adult carrying a compound heterozygote for IVS-Ⅱ-55 (T→G) and βCD27-28M/N were 65.0 fL, 20.3 pg and 5.8% respectively. The hematological phenotype index (MCV, MCH, HbA 2 and HbF) of one adult carrying a compound heterozygote for IVS-Ⅱ-55 (T→G) and SEA-HPFH were 81.9 fL, 26.5 pg, 3.0% and 29.0% respectively. The HbA 2 levels of all carriers of IVS-Ⅱ-55 (T→G) heterozygote alone were in normal range. No abnormal hemoglobin band was detectable on hemoglobin electrophoresis for all the carries. @*Conclusion@#The carriers of IVS-Ⅱ-55(T→G) heterozygote alone were asymptomatic. The phenotype of compound heterozygote for β-thalassemia was similar to that of β-thalassemia alone.

The effect of membrane protein NMHC ⅡA on invasion and metastasis of bladder cancer cells / 国际检验医学杂志

Objective Non-muscle myosin heavy chain ⅡA (NMHC ⅡA ) plays a significant role in tumor progression and metastasis .Our prior study showed that the expression of NMHC ⅡA was much higher in human bladder cancer sample than that in adjacent tissue .The increased level of NM HC ⅡA expression was correlated with worse prognosis .However ,the role of NMHC ⅡA is unknown in the invasion and metastasis of bladder cancer .Methods RT-PCR and western blotting were used to examine NMHC ⅡA expression lev-els in normal bladder epithelial cells and bladder cancer cell lines .T he migration and invasion ability of cells was tested by wound healing assay and Transwell invasion assay ,respectively .Results Our study showed that knockdown of NMHC ⅡA inhibited migration and invasion in bladder cancer cell line .Conclusion The study indicated that NM HC ⅡA expression increased the invasion and metastasis ability of bladder cancer cell line in vitro .

The screening and validating of PLCE1 as a new gene related to the pathogenesis of sporadic colorectal cancer / 中华普通外科杂志

Objective To screen for and validate unknown tumor suppressor genes (TSGs) in sporadic colorectal cancer (CRC) patients.Methods Through loss of heterozygosity (LOH) analysis on chromosome 10 in sporadic CRC,we have found D10S185 (10q23.31-24.33 ) exhibit a higher LOH frequency in our previous study.In present study,we screen for unknown TSGs in this region through the microarray.The expression of the new gene was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR).RT-PCR,immunohistochemistry and Western blot were done in colorectal cancer tissues with their pair-matched normal tissues in 50 cases to validate the results of microarray.Results Through the microarray-based high throughput screening,we found 4 significant down-regulated genes:PLCE1,CPEB3,NKX2-3 and SEMA4G,among them the down-regulation of PLCE1 was most significant.The results of qRTPCR were in relative agreement with the DNA microarray data.RT-PCR,immunohistochemistry and Western blot also showed that the expression of PLCE1 was at low levels in 46％ cancer tissues compared with normal tissues,more frequent in the poor differentiation tumor in patients under age 60 years (P ＜ 0.05 ).Conclusions This study demonstrated that down-regulation of PLCE1 was related to the tumorigenesis of sporadic colorectal cancer.PLCE1 might play a suppressive role in the development of colorectal cancer.

Effects of stably silencing FOXM1 by shRNA on cell growth of hepatocellular carcinoma in vitro / 中华普通外科杂志

Objective To evaluate the effect of sustained silencing Forkhead box Ml (F0XM1) gene by short-hairpin RNA (shRNA) expression vector on cell growth of hepatocelluar carcinoma (HCC) in vitro.Methods Four shRNA expression vectors targeting different sequences of human F0XM1 mRNA were constructed.The expression vector with the best interfering effect and the negative control plasmid were used to transfect HCC cell line QGY-7703, stably transfected cell clones were selected by neomycin (G418).Cell growth was evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and colony formation was assessed by clonogenic assay.Cell apoptosis was detected by double staining with APC conjugated Annexin V and PI.Results F0XM1 protein was detected with different levels in all these studied human cell lines.The expression vector shRNA-1026 exhibited excellent interference effect after transient transfection, which showed 38.5% and 53.2% reduction of FOXM1 mRNA and protein level respectively.The growth of QGY-7703 cells was inhibited after stable inhibition of FOXM1 expression by shRNA-1026, which was indicated by decreased absorbance value of the test group after culture for 48, 72 and 96 h compared to control group (t = 10.830,3.578 and 5.734 respectively, P ＜ 0.05).Stable inhibition of F0XM1 also led to reduced colony formation ( t = 5.336, P ＜ 0.05 ) and increased apoptosis of QGY-7703 cells in comparison to control cells (t = 6.827, P ＜ 0.05 ).Conclusions Stable silencing F0XM1 gene by shRNA suppresses the growth of HCC cells in vitro.

Effects of HGF in Celsior solution on levels of INF-γ. IL-4 and IL-10 in a rat liver transplantation model / 中华肝胆外科杂志

Objective To explore the impact of recombinant human hepatocyte growth factor (rhHGF) in Celsior (CS) solution on the expression of INF-γ, IL-4 and IL-10 in a rat liver transplan-tation model. Methods After flushed with CS solution with addition of rhHGF (experimental group) or saline (control group), NHBD livers were stored at 4℃; for 16 h.then they were transplanted using the two-cuff technique with arterial reconstruction. The serum levels of INF-γ, IL-4 and IL-10 at lh after reperfusion were detected using ELISA. The INF-γ, IL-4 and IL-10 mRNA in the corresponding liver tissue were determined by RT-PCR. The 7-day survival rate was calculated and the histopatho-logical examination results were analyzed by hematoxylin and eosin staining. Results Compared with the control group, the experimental group showed lower INF-γ level and higher IL-4 and IL-10 levels in serum at 1 h after reperfusion (P<0. 05). The level of INF-γ mRNA in liver tissue was significant decreased at 1 h after reperfusion (P<0. 05) , and the level of IL-4 and IL-10 mRNA was significantly increased in the experimental group (P<0. 05). In experimental group, recipients got a better survival rate and histopathological examination showed a well-preserved hepatic architecture without hepatocyte necrosis, milder sinusoidal and portal congestion. Conclusion Adding exogenous rhHGF in CS solu-tion can protect NHBD livers from ischemia-reperfusion injury and prolong the survival in rats, which might be due to down-regulation of TNF-γ and up-regulation of IL-4 and IL-10.

Loss of heterozygosity of chromosome 20 in sporadic colorectal cancer / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To analyze the loss of heterozygosity (LOH) of chromosome 20 in patients with sporadic colorectal cancer to identify additional loci involved in colorectal tumorigenesis.</p><p><b>METHODS</b>Polymorphic microsatellite markers were analyzed in 83 colorectal cancer patients' tumor and normal DNA by PCR. PCR products were electrophoresed on an 377 DNA sequencer. Genescan 2.1 and Genotype 2.1 software were used in the LOH scanning and analysis. Comparisons between LOH frequency and clinicopathological data were performed by chi(2) test. P < 0.05 was considered statistically significant.</p><p><b>RESULTS</b>The average LOH frequency in the long arm, short arm and whole chromosome 20 was 21.1%, 26.7% and 22.8%, respectively. Chromosome 20 exhibited relatively high LOH frequency, particularly in the regions of 20p and 20q11.1-q13.1.</p><p><b>CONCLUSION</b>There is notable genetic instability on chromosome 20 in sporadic colorectal carcinoma patients; that is, mutation on chromosome 20 is closely associated with sporadic colorectal carcinogenesis. Also, there may be tumor suppressor genes related to sporadic colorectal carcinoma near the region 20q11.1-q13.1.</p>

Technical improvement of "two-cuff method" of orthotopic liver transplantation in rats / 中国普通外科杂志

ObjectiveTo investigate an improved technique of orthotopic liver transplantation with the ("two-cuff method") in rats, and to observe allograft rejection after transplantation. Methods30 Wistar rats were grafted with livers of SD rats and 30 SD rats were grafted with livers of SD rats by using a modified "two-cuff technique" and examined for postoperative rejection. ResultsThe anhepatic period was about 15 minutes. The 24-hour survival rate was 100%. The Wistar rats grafted with SD livers died 8 to 15 days (after) liver transplantation, and the histopathologic examination showed various degrees of allograft rejection. The SD rats grafted with livers of SD rats had a survival rate of 97% at 3 weeks postoperatively. ConclusionsThe modified "two-cuff technique" can effectively reduce operative time, decrease postoperative (complications) and increase the survival rate of othortopic liver transplantation in rats. The transplantation model of Wistar rats grafted with livers of SD rats can be used to study allograft rejection.

Apoptosis and Fas System Are Significantly Involved in the Process of Liver Cirrhosis Converting into Hepatocellular Carcinoma / 华中科技大学学报（医学）（英德文版）

To investigate the roles of apoptosis and the Fas system (Fas, Fas ligand, soluble Fas) in the process of liver cirrhosis (LC) converting into hepatocellular carcinoma (HCC), expression of Fas and Fas ligand (FasL) in 49 LC and 36 HCC samples was detected by immunohistochemical method. Apoptosis was detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) method. Serum soluble Fas (sFas) levels in 28 cases of LC and 27 cases of HCC were measured by enzyme-linked immunosorbent assay (ELISA) method. Compared with LC, apoptotic indices (AI) in HCC tissues were significantly reduced (P<0.001), expression of Fas was decreased (P<0.05), and that of FasL was increased (P<0.05). Serum sFas levels in HCC patients were significantly higher than those in normal controls. Down-regulation of Fas expression, up-regulation of FasL expression in hepatocytes and elevation of sFas level in serum might contribute to tumor escape from immune surveillance of the body. Apoptosis and the Fas system are significantly involved in the process of liver cirrhosis converting into hepatocellular carcinoma.

Apoptosis and Fas System Are Significantly Involved in the Process of Liver Cirrhosis Converting into Hepatocellular Carcinoma / 华中科技大学学报（医学）（英德文版）

To investigate the roles of apoptosis and the Fas system (Fas, Fas ligand, soluble Fas) in the process of liver cirrhosis (LC) converting into hepatocellular carcinoma (HCC), expression of Fas and Fas ligand (FasL) in 49 LC and 36 HCC samples was detected by immunohistochemical method. Apoptosis was detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) method. Serum soluble Fas (sFas) levels in 28 cases of LC and 27 cases of HCC were measured by enzyme-linked immunosorbent assay (ELISA) method. Compared with LC, apoptotic indices (AI) in HCC tissues were significantly reduced (P<0.001), expression of Fas was decreased (P<0.05), and that of FasL was increased (P<0.05). Serum sFas levels in HCC patients were significantly higher than those in normal controls. Down-regulation of Fas expression, up-regulation of FasL expression in hepatocytes and elevation of sFas level in serum might contribute to tumor escape from immune surveillance of the body. Apoptosis and the Fas system are significantly involved in the process of liver cirrhosis converting into hepatocellular carcinoma.