ObjectiveTo establish a simple and sensitive method to detect MPL515 mutations in peripheral blood of ET and PMF patients,and investigate the frequencies of the MPL515 and JAK2V617F mutations in Chinese patients.MethodsTotallv 261 patients of ET and 25 PMF cases were collected from Huashan Hospital of Fudan University and DNA samples were isolated from peripheral blood of these cases.SYBR GreenⅠreal-time PCR was used to detect JAK2V617F mutation.Taqman probe was designed to be specific for the three types of mutations ( MPl515wt,MPLW515L and MPIW515K).Real-time PCR was used to detect MPL515 mutations.Tbe results were confirmed by sequencing after T-A cloning.Results Among 261 ET patients,119 cases (45.6％ ) were identified as JAK2V617F mutation carriers and 7 cases (2.7％ ) were detected to be MPl515 mutation carriers,including 5 cases with MPLW515L,1 case with MPLW515K and 1 ease with MPLW515L + K.Additionally 10 cases with JAK2V617F(40.0％ ) and 3 cases with MPL515 ( 12.0％ ) were screened out in 25 PMF patients,including 1 case with MPLW515L and 2 cases with MPLW515L + K.One ET patient was found to harbor concurrent JAK2V617F and MPL515 mutations.ConclusionJAK2V617F mutation is the major molecular marker of ET and PMF,meanwhile MPL515 mutation is important and useful complement.

Objective To investigate a new single nucleotid polymorphism (SNP) intron5(+4668C/T) in SLC22A12 in primary gout patients and the association between clinical characteristics and genotypes. Methods One hundred and one primary gout patients and 186 healthy subjects were recruited into this study. Blood pressure, body mass index (BMI) was recorded. Serum uric acid, glucose, lipid and creatinine were detected. DNA was extracted from peripheral blood to amplify the fragment located in intron 5. The genotypes of SLC22A12 can be detected with high-resolution melting (HRM) assay, followed by sequencing analysis. Chi-square test was used for statistical analysis. Results ① A new SNP in intron 5 of SLC22A12 was identi-fied successfully by HRM, which was defined as intron 5 (+4668C/T). CC, CT and TT genotypes were unam-biguously distinguished with HRM technology, which was fully concordant with sequencing. ②The genotypes of CC, CT and TT in male and female groups were 28.1%, 33.7%, 38.2% and 20.0%, 47.1%, 32.9%, respectively.③ However, no significant differences of genotype distribution were found concerning BMI, blood pressure, creatinine, total cholesterol and triglyceride in both male group and female group. But the serum uric acid levels in the CC genotype were significantly higher than those with the CT+TT genotypes. ④ The genotype frequencies of CC and CT+TT in high uric acid group were remarkably different from those in low uric acid group (21.2%, 78.8%,; 35.0%, 65.0%; P<0.05). Conclusion A new SNP has been successfully discovered with HRM technology with simplicity, rapidity and accuracy. T allele of intron 5 (+4668C/T) may be a genetic protective factor for hyperuricemia among Chinese population.

Objective To explore the expression of basic fibroblast growth factor(bFGF) in pulmonary tissues of rats with bleomycin-induced pulmonary fibrosis and the role of bFGF in vascular injury.Methods Pulmonary fibrosis was induced in rats by intratracheal injection of bleomycin(BLM).Lung expression of bFGF proteins was assessed by SABC immunohistochemistry using bFGF monoclonal antibody and quantitative image analysis.Results The expression of bFGF in the lung tissues of BLM group was mainly distributed in bronchiole epithelial cells,muscular layers of bronchi and arterioles,and alveolar macrophage.It was higher than that in control group,with significant difference(P

Objective According to the requirements of the College of American Pathologists (CAP),to validate the laboratory developed test-JAK2 V617F mutation.Methods This is a clinical laboratory diagnosis study.Fifty-seven peripheral blood and bone marrow samples were collected from the BCR/ABL-negative myeloproliferative neoplasms (MPN) patients in Huashan Hospital from Apr 2010 to Dec 2010.JAK2 V617F mutation was detected by fluorescence PCR combined with melting curve analysis.The accuracy,precision,analytical sensitivity,and anti-interference ability of the assay were validated.The Kappa test was used to evaluate accuracy.Coincidence rate was used to evaluate the repeatability.Paired t test was used to evaluate analytical sensitivity.Results There was a close agreement between the reference method(sequencing) and the test method (fluorescence melting curve analysis) (Kappa =0.89).The coincidence rate was 100％.The results of the assay was not affected by lipoprotein (＜ 24 mmol/L) or bilirubin (＜450 μmol/L).The detection limit of the assay was 5％.Conclusion JAK2 V617F mutation assay by fluorescence PCR combined with melting curve analysis could be applied to the clinical laboratory.

Objective To establish a single-tube detecting system for the simultaneous identification of JAK2 V617F and JAK2 exon12 mutations.Methods Genomic DNA of cell line PC-3 was utilized as the wild type control,while genomic DNA of cell line HEL and plasmids with diverse JAK2 exon 12 mutations were used as the positive controls for JAK2 V617F and exon12 mutations.Multiplex PCR was performed to amplify the different amplicons combined with high-resolution melting (HRM) analysis,which established the multiplex detecting system for JAK2 V617F and exon12 mutations.Meanwhile 42 cases of polycythemia vera patients were collected to detect 2 kinds of JAK2 mutations by the above system and routine methods.Results The multiplex JAK2 mutations detecting system was successfully established by multiplex PCR combined with high-resolution melting curve analysis,which could simultaneously detect JAK2 V617F and JAK2 exon12 mutations.The analytical sensitivities of 2 mutations in this system were both up to 5％ and the precision (coefficient of variation) of intra-and inter-assay of the melting temperature (Tm) of 2 amplicons were separately less than 0.01％.37 cases were identified JAK2 V617F mutations from 42 polycythemia vera patients,while 2 JAK2 exon12 mutations cases were found from 5 JAK2 V617F negative patients.Compared with routine methods,the results matched the rate of 100％.Two cases of JAK2 exon 12 mutations were confirmed to the mutation types of H538K539delinsL and F537-I546dul10 + F547L by cloning and sequencing.Conclusions This method can simultaneously detect two kinds of JAK2 mutations in the peripheral blood and will contribute to the molecular diagnosis of myeloproliferative neoplasms,especially polycythemia vera.

Objective To evaluate the sensitivity, repeatability and accuracy of microarray digital PCR system in detecting JAK2 V617F mutation, which was closely related to myeloproliferative neoplasms (MPN).Methods All of the 31 MPN patients with JAK2 V617F mutation, including 18 cases of polycythemia vera(PVs),11 primary thrombocythemias (ETs) and 2 primary myelofibrosis (PMFs), were collected from Huashan Hospital, Fudan University during 2014 -2015, while 10 normal controls and 6 cases with abnormal increased hemoglobin were involved.Human erythroleukemia cell line ( HEL ) and colorectal cancer cell SW480 were used as the mutant and the wild type control, respectively.The sensitivity of microarray digital PCR were verified by detecting the gradient diluted mutation standard harboring 30%, 10%, 1%, 0.1%and 0.01%mutant allele burden, respectively .Repeatability was evaluated by detecting 1%and 10% mutated samples for 5 times, respectively.MGB probe real time PCR was selected as the reference method to verify the accuracy of the digital PCR.Results With digital PCR, the accurate quantitation of JAK2 V617F mutation was achieved down to 0.1%, which is approximate to 0.16 copies per microliter.The results obtained from the two kinds of technique showed a high correlation by linear regression analysis (R2 =0.998 3).The results of repeated samples showed CVs as 17.18% for 1%mutant allele burden and 7.50%for 10%.Among all cases, the 31 patients known mutated were detected as positive and 10 controls as negative by both digital PCR and Real time PCR.In another 6 cases, 2 were found JAK2 V617F mutation of low allele burdens of 0.37% and 0.18% by digital PCR but detected as negative by real time PCR.Conclusions Microarray digital PCR offers a higher sensitivity and better repeatability than real time PCR which could help detect rare JAK2 V617F mutations in MPNs accurately.

ObjectiveTo investigate the expression levels of forkhead box A2 (FOXA2) and forkhead box J2 (FOXJ2) in hepatocellular carcinoma (HCC) tissue and the association of FOXA2 and FOXJ2 with HCC. MethodsClinical data and pathological tissue samples were collected from 54 patients with pathologically confirmed HCC in The First Affiliated Hospital of Henan University of Traditional Chinese Medicine from January 2014 to July 2019. The immunohistochemical SP method was used to measure the protein expression levels of FOXA2 and FOXJ2 in HCC tissue, and their association with HCC-related clinicopathological features and patient prognosis was analyzed. The chi-square test and the adjusted chi-square test were used for comparison of categorical data; a Spearman correlation analysis was performed to investigate the correlation between the expression of FOXA2 and FOXJ2; the Kaplan-Meier method was used for survival analysis; Image-Pro Plus was used to perform the semi-quantitative analysis of the expression of FOXA2 and FOXJ2; the Wilcoxon rank-sum test was used for comparison between groups. ResultsThe positive rates of the protein expression of FOXA2 and FOXJ2 in HCC tissue were 70.37% (38/54) and 75.92% (41/54), respectively, and there was a significant positive correlation between the expression levels of FOXA2 and FOXJ2 (rs=0.648, P＜0.001). In both negative and positive groups, the expression level of FOXA2 was associated with tumor diameter, degree of tumor differentiation, number of tumors, and alpha-fetoprotein (χ2=5.440, 4.113, 4.352, and 3.865, P=0.020, 0.043, 0037, and 0.049), and the expression level of FOXJ2 was associated with the degree of tumor differentiation (χ2=9.267, P=0.002). The group with positive expression of FOXA2 and FOXJ2 had a significantly higher cumulative survival rate than the group with negative expression of FOXA2 and FOXJ2 (P＜0.01). ConclusionThe expression levels of FOXA2 and FOXJ2 are associated with the development, progression, and prognosis of HCC, and they have a synergistic effect in the development and progression of HCC.

Objective To illustrate if the racial coefficient (Rc) be biased by different reference GFR (rGFR) distribution among studies. Methods 1405 white and 321 African American participants in MDRD study and 684 chronic kidney disease(CKD)patients in Chinese eGFR Investigation Study were included.Firstly.the unweighted datasets of white and Chinese were stacked together.rGFR,age and plasma creatinine (Pcr) were log transformed.Linear regression model was constructed using log transformed rGFR as dependent,gender,race and log transformed Pcr and age as independent.Unweighted RC (uRC) for Chinese was calculated.Then.the Chinese CKD distribution of rGFR was weighted to be the same as that in White American.and weighted RC (wRC)for Chinese was calculated.The cases of White.and African-American were stacked together.The cases of African-American were weighted to make the rGFR distribution the same as that in White-American and Chinese population respectively,and RCs for African-American were calculated. Resuits The uRC for Chinese was 1.197(1.180-1.211)and the wRC was 1.130 (1.117-1.143).The two RCs did not overlap with each other.The RCs for African-American were 1.205(1.19-1.219)and 1.233(1.219-1.247)respectively. Conclusions The RCs were influenced by the difference of rGFR distribution.To find out the real RC.an intemational collaborative study iS needed,with the same rGFR measure method.strict control of Pcr measurement,and the same rGFR distribution.

Objective To test and evaluate the JAK2 gene V617F mutation in patients with myeloproliferative tumors based on i-densy IS-5320 platform according to ISO15189 accreditation requirements.Methods Instrument performance verification.Selected from December 2014 to February 2017, 20 cases of JAK2 V617F mutation positive peripheral blood samples from Huashan Hospital of the Shanghai FuDan University Medical College and 20 cases of peripheral blood samples with negative JAK2V617F mutation.The Realtime PCR with TaqMan MGB probe was selected as the control method to verify and evaluate the accuracy of testing JAK 2 V617F mutation on i-densy IS-5320 platform.Whole blood samples were used to evaluate the reproducibility , cross-contamination and anti-interference ability of this platform.The ability of mutation load was verified by detecting mixtures of human erythnoleukemia cells and colorectal cancer cell HCT116 with 12 different proportions.Results I-densy IS-5320 platform and TaqMan MGB probe real-time fluorescence quantitative PCR show the same result .The within-run reproducibility and between-run reproducibility are both 100%.There is no observed contamination .High bilirubin and high triglyceride blood samples have no obvious interference on mutation detection .The mutation ratio with a load as low as 0.25%could be tested stably by i-densy IS-5320 platform.The detecting peak of melting curve can reflect the ratio of JAK2 V617F mutation to some extent.Conclusions I-densy IS-5320 can detect the mutation of JAK2 V617F gene in the whole blood directly.It has high sensitivity, accuracy and stability, and is easy to operate, and also can reflect the mutation load of JAK2 V617F, which could meet the clinical requirements for the detection of mutations.

Treatment regimens for primary liver cancer mainly include surgical resection, liver transplantation, local treatment, and systemic treatment, but there are still problems such as easy metastasis, easy recurrence, and poor prognosis. Most patients are already in the advanced stage at the time of diagnosis and thus lose the opportunity for surgery and require multidisciplinary combined treatment. Traditional Chinese medicine (TCM) has unique advantages in the treatment of primary liver cancer and can exert a synergistic effect in collaboration with different Western medicine treatment methods. This article establishes TCM treatment regimens based on the different clinical stages of primary liver cancer, i.e., surgical resection for early-stage liver cancer to strengthen vital Qi and prevent recurrence, minimally invasive treatment of middle-stage liver cancer to eliminate cancer toxicity and improve efficacy, and symptomatic treatment of advanced liver cancer to reduce pain and maintain quality of life.