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Objective:To explore the targeted relationship between the CXC chemokine ligand 10 (CXCL10) and miR-34c-5p in invasive ductal carcinoma of breast.Methods:A total of 56 cases of invasive ductal carcinoma of breast and 14 cases of benign breast tissues surgically resected and pathologically confirmed in the Third Affiliated Hospital of Sun Yat-sen University were collected from March 2016 to March 2017. The expressions of CXCL10 and miR-34c-5p in the two groups were detected using HTA2.0 and miRNA4.0 chips and verified by reverse transcription quantitative real-time PCR (RT-qPCR). The correlation between the two expressions was analyzed using Pearson correlation analysis and the targeted relationship was predicted by IPA software. The dual luciferase reporter gene system was used to verify the targets of the two and finally verified in breast cancer cell lines MCF-7 and ZR-75-30.Results:The results of gene microarray showed that the mRNA expression values of CXCL10 in invasive ductal carcinoma of breast and benign breast tissues were 7.135±1.350 and 4.348±0.722, and the mRNA expression values of miR-34c-5p in the two groups were 1.309 (1.001, 2.055) and 3.948 (3.278, 4.371), with a statistically significant difference ( t=7.628, P<0.001; Z=-4.405, P<0.001). Pearson correlation analysis showed that the mRNA expressions of CXCL10 and miR-34c-5p were negatively correlated ( r=-0.327, P=0.004). The expression of CXCL10 mRNA was related to the expressions of human epidermal growth factor receptor-2 ( t=2.415, P=0.019) and Ki-67 ( t=2.483, P=0.016), and the expression of miR-34c-5p mRNA was related to the histological grading ( χ2=8.626, P=0.013) and estrogen receptor/progesterone receptor ( Z=-2.195, P=0.028) in patients with invasive ductal carcinoma of breast. RT-qPCR showed that the relative expressions of CXCL10 mRNA in invasive ductal carcinoma of breast and benign breast tissues were 6.059±1.714 and 1.000±0.232, the relative expression levels of miR-34c-5p in the two groups were 0.093±0.004 and 1.000±0.244, with statistically significant differences ( t=3.484, P=0.002; t=5.112, P<0.001), which was consistent with the results of microarray. The results of double luciferase reporter gene showed that, after overexpression of miR-34c-5p, the luciferase activities of WT-CXCL10 and MUT-CXCL10 were 1.117±0.040 and 1.647±0.031 in MCF-7 cells, and the luciferase activities of them were 0.885±0.051 and 1.430±0.020 in HeLa cells, with statistically significant differences ( t=18.120, P<0.001; t=24.040, P<0.001). After transfection with miR-34c-5p and miR-NC respectively, the relative expression levels of CXCL10 were 0.008±0.000 and 0.012±0.000 in ZR-75-30 cells, and the relative expression levels of CXCL10 were 0.315±0.000 and 0.386±0.004 in MCF-7 cells, with statistically significant differences ( t=20.384, P<0.001; t=3.473, P=0.026). Conclusion:The expression of miR-34c-5p in invasive ductal carcinoma of breast is down-regulated, while the expression of CXCL10 is up-regulated. CXCL10 is the target gene of miR-34c-5p.
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Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma.Generally,it can be divided into two immunological subgroups:germinal center B-cell-like (GCB)and non-germinal center B-cell-like (non-GCB),and they are different both in distribution and prognosis in different populations.And DLBCL has unique genetic characteristics in Chinese populations.With the global advances in personalized medicine,people gradually realize that only personalized therapy could be obtain maximal therapeutic effect for different populations,especially person from different kind of ethnic group.
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Objective To investigate the types,antimicrobial resistance,and disinfectant resistance of pathogens isolated from hospital environmental inanimate surfaces and hands of health care workers (HCWs).Methods Pathogens isolated from hospital environmental inanimate surfaces and hands of HCWs in intensive care units and general wards in 16 hospitals in Beijing were performed bacterial identification,antimicrobial susceptibility testing,and disinfectant re-sistance testing. The carriage of antimicrobial resistance genes and disinfectant genes in pathogens were also detec-ted.Results A total of 979 specimens were collected from inanimate surfaces and hands of HCWs in 16 hospitals,75 (7.66% )pathogenic strains were isolated,78.67% of which were gram-negative bacilli. The top 3 pathogens were Pseud-omonasaeruginosa (P.aeruginosa,n= 24),Enterobactercloacae (E. cloacae,n= 14),and Klebsiella pneumoniae (K. pneumoniae,n= 4 ). One P. aeruginosa strain was resistant to aztreonam,gentamycin,tobramycin,ciprofloxacin,and levofloxacin;One E. cloacae strain was resistant to piperacillin,7 strains were resistant to nitrofurantoin;4 K. pneumoni-ae strains were all resistant to piperacillin,2 were resistant to cephalosporins,and 1 was resistant meropenem. P. aerugi-nosahad7drug-resistantgenes,positiverateofmirwas100.00% ;E.cloacaehad4drug-resistantgenes,positiveratesof tem 1and shv were both 100.00% ;K. pneumoniae had 5 drug-resistant genes,positive rates of shv and mir were both 100.00% . The resistant rates of P. aeruginosa and E. cloacae to chlorhexidine gluconate were 4.17% and 57.14% re-spectively,to trichloroisocyanuric acid were both 50.00% ,positive rates of drug-resistant genes (qacE△1-sul 1)were 79. 17% and 57.14% respectively;K. pneumoniae had no resistance to two kinds of disinfectant,dug-resistance gene was not found.Conclusion Multiple common pathogens which can cause healthcare-associated infection exist in hospital environ-mental inanimate surfaces and hands of HCWs,which are dominated by gram-negative bacilli,pathogens had resistance to antimicrobial agents and disinfectant in different degrees.
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Objective To develop a sensitive and stable substrate for enzyme‐linked immunosorbent assay(ELISA)and carry out methodology evaluation tests on it .Methods A kind of substrate with tetramethyl benzidine and urea peroxide as its main compo‐nents were made up ,its sensitivity ,precision and storage stability were evaluated and compared with other three kinds of substrate . Results This substrate was more sensitive than two of the three commercial substrates .The variable coefficient of precision test was 3 .5% .The optical densities(OD) of the new substrate after stored one day ,seven days ,one month ,six months ,twelve months in 4 ℃ environment were 2 .268 、2 .403 、2 .358 、2 .278 、2 .330 respectively ,the standard deviation was 0 .056 185 ,the variable coeffi‐cient was 2 .4% .Conclusion The substrate proposed in this paper displays good sensitivity ,precision and stability .The preparation method is simple ,reproducible .This substrate not only satisfies the requirements of laboratory research ,but also meets with the de‐mands for commercial kit development due to its function as an important component of test kits .It is an economical and effective choice for both laboratories and research transformation .
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Objective To explore the mechanism and therapeutic effects of wet dressing with 50％ magnesium sulfate ( MgSO4 ) as a part of comprehensive treatment for wound resulted from pit viper bites in children.Methods A total of 61 children with pit-viper-bitten wound enrolled from May 2009 through May 2011 were divided into two groups,namely the comprehensive treatment group (n =31 cases) and the conventional treatment group (n =30 eases). In the comprehensive treatment group,50％ magnesium sulfate wet dressing applied to the swelling parts of body in the early stage in addition to the conventional treatment and topically bandaged once a day keeping wet with spraying saline on the top of dressing.Before the treatment and on the 1st day,the 3rd day and the 5th day of treatment,the values of myocardial enzyme and the circumference of swelling parts were recorded,and the differences were analyzed by t- test.The differences in rate of wound healing in one week between two groups were analyzed by chi-square test.Results Before the treatment,there was no statistical difference in clinical features between the two groups with different modes of treatment for the viper-bitten children (P ＞ 0.05 ). During comprehensive treatment,the values of myocardial enzyme and the circumference of swelling parts decreased more markedly than those in conventional treatment group ( P ＜ 0.05 ).The rates of wound healing in one week of two groups were 80.5％ and 56.6％ respectively.Conclusions The comprehensive treatment with adjuvant of 50％ MgSO4 for the viper-bitten children can effectively reduce the tissue damage caused by venom,and the swelling as well,restoring function of body and shortening the time for wound healing and preventing complications.
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There is a high prevalence of thalassemia in the South of China. To explore the genotype of alpha-thalassemia as well as the distribution of alpha globin gene mutation in the South of China, 356 patients with heterozygote alpha(+) thalassemia, heterozygote alpha(0) or homozygote alpha(+) thalassemia and 78 patients with HbH were analyzed. The gene diagnosis methods including Gap-PCR, nested-PCR, PCR-RE, PCR-SSCP, 4P-ASPCR and DNA sequence analysis were used. The results showed that among 356 patients, 295 patients with --SEA/alphaalpha (82.87%), 1 patient with alphaalpha/alpha-alpha(3.7) (0.28%), 3 patients with alphaalpha/alpha-alpha(4.2) (0.84%), 3 patients with alphaalpha/alpha(CS)alpha (0.84%), 1 patient with alphaalpha/alphaalpha(QS) (0.28%) and 2 patients with alphaalpha/alpha(Westmead) alpha (0.56%) were found. The homozygote with -alpha(4.2) or -alpha(3.7) was not found. In 78 patients with HbH, 29 patients with --SEA/alphaalpha(-3.7) (37.2%), 20 patients with --SEA/alphaalpha(-4.2) (25.6%), 19 patients with --SEA/alphaalpha(CS) (24.3%), 2 patients with --SEA/alphaalpha(QS) (2.6%) were detected, and other remaiming 8 patients were needed to be defined. Among the non-defined 8 patients, the synonymous mutation with C-->G transversion (GCC-GCG) at codon 65 in the exon 2 of alpha 2-globin gene was detected in 2 unrelated HbH patients came from Guangxi province. Whether it correlated with the phenotype of HbH disease or it is only a single nucleotide polymorphism site (SNPs), should be confirmed in the future. In addition, a set of gene diagnosis methods based on PCR to screen deletion and non-deletion genotypes of alpha-thalassemia in Chinese was improved. A new method, 4P-ASPCR, to detect Hb CS and Hb QS was also developed. The method was verified to be more accurate, time-saving and economic. In conclusion, the genotypes of alpha-thalassemia in Chinese are very complicated, the genotypes of alpha-thalassemia in Chinese need to be further studied, the results of this research probably have practical significance for the gene diagnosis or antenatal diagnosis of alpha-thalassemia in the South of China.
Subject(s)
Humans , Base Sequence , China , DNA , Chemistry , Genetics , DNA Mutational Analysis , Gene Deletion , Gene Frequency , Genotype , Globins , Genetics , Hemoglobin H , Genetics , Hemoglobins , Genetics , Hemoglobins, Abnormal , Genetics , Molecular Sequence Data , Mutation , Polymorphism, Single-Stranded Conformational , alpha-Thalassemia , Genetics , PathologyABSTRACT
Objective To develop a rapid method based on ARMS, named four primers allele-specific PCR(4p-AS PCR), to detect the most common non-deletional ?-thalassemia mutation Hb Constant Spring(Hb CS) by PCR technique. Methods The 4p-ASPCR was used to detect the Hb CS mutation of ?-thalassemia in 38 DNA samples of Hb H disease patients and using PCR-RE and DNA sequencing to confirm the results. Results Among the 38 Hb H disease patients 15 cases was revealed to carry Hb CS, 16 cases were deletional Hb H, and 7 cases need to be defined.Conclusion A simple, rapid and reliable method, named 4P-ASPCR, to detect Hb CS mutation have been developed. It is also may be useful in screen other point mutations such as Hb Quong Sze.
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AIM: The purpose of this study was to explore the expression of estrogen receptor(ER) and progesterone receptor(PR) mRNA in endometrium of rats with endometriosis. METHODS: The rat model of endometriosis was established, and the expression of ER, PR mRNA in the endometrium was examined by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The expression of ER and PR mRNA in ectopic endometrium was significantly lower than that in eutopic and normal endometrium (P
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AIM: To clone human ?-globin gene carrying a thalassemic mutation IVS II654(C→T) and establish a eukaryotic expression system for high-level expression of human ? IVS II654 gene in mouse erythroleukaemia(MEL) cells. METHODS: The fragments of human ? 654 gene isolated from the ? thalassemia patients homozygous for the ? 654 mutation were amplified by PCR, and cloned to plasmid pBGT51. Then, the human ? LCR and ? 654 gene were subcloned from plasmid pBGT51 to the stable mammalian expression vector pcDNA3.1+ together, and the MEL cells were transfected with this vector using commercially available cationic lipid FuGENE6. The MEL cells were induced for further maturation by DMSO and the expression of human ? 654 gene in the MEL cells was identified by RT-PCR. RESULTS: A mammalian expression system of human ? thalassemic mutation ?IVS II654(C→T) was established. CONCLUSION: The level and the reliability of expression of human ? 654 gene in the MEL cells in vitro are similar to that in vivo in human body. This may be a valuable gene therapy model for human ? thalassemic mutation ?IVS II654(C→T).