Objective To establish real-time PCR method for the detection of JAK2 V617F mutation and evaluate its clinical significance in patients with myeloprollferative disorders and leukemia.Methods 71 chronic myelocytic leukemia(CML) patients, 22 essential thrombocythemia (ET) patients, 11 primary myelofibrosis (PMF) patients, 9 polycythemia vera (PV) patients and 7 cosinophilia patients were enrolled in this study. JAK2 V617F mutation was determined by real-time PCR and amplification refractory mutation system (ARMS), followed by sequencing. Human erythroleukemia cell (HEL cell)DNA was used as homozygous control of JAK2 V617F mutation. The detection limit for either real-time PCR or ARMS was evaluated. Results Real-time PCR assay showed that there was a melting temperature(Tin) peak at (75.0±0.2)℃ for wild type samples and a Tm peak at (76.6±0.2)℃ for mutation type samples. JAK2 V617F mutation was detected in 8(88.9%) patients with PV, 12(54.5%) patients with ET and 7(63.6%) patients with PMF respectively. But there was only one positive case in 71 CML patient (1.4%). The results showed complete concordance with ARMS results and confirmed by sequencing. The mutation could be detected in 102 HEL cells per 106 white blood cells by real-time PCR, whereas the mutation can be assessed in 104 HEL cells per 106 white blood cells by ARMS. Thus, the sensitivity of real-time PCR was 100-fold higher than ARMS. Conclusions The real-time PCR method is successfully established for detection of JAK2 V617F mutation. This method is more sensitive, convenient than ARMS, and suitable for clinical application. There is high frequency of JAK2 V617F mutation in myeloproliferative disorders and it could be used as the diagnostic marker for myeloproliferative disorders.

Objective To explore immunological mark and the outcome of disease after pharmacological treatment in human NCC with praziquantel. Methods 35 patients were treated with praziquantel for 6 months. Levels of serum IL-2,IFN-γ, IL-12, IL-5, IL-10 and TNF-α were measured before and after treatment. Results Th1 type cytokines IL-2, IFN-γand IL-12 were up regulated after treatment( P ＜ 0. 01 ) . Levels of TNF-α and Th2 type cytokines IL-5 and IL-10 were remarkable decreased after treatment( P ＜ 0. 01 ). The levels of IL-2, IFN-γ and IL-12 from the patients with good response to the treatment is higher than those with no response to the treatment. Conclusion Th1 type cytokines were up regulated while Th2 type cytokines were down regulated in the NCC patients treated with praziquantel. The protective immunity may be related to the Thl cell activation.

Objective To investigate the relationship between HLA-B * 5801 allele and severe cutaneous adverse reactions caused by allopurinol or other drugs.The clinical value of HLA-B * 5801 as the marker of allopurinol-SCAR was evaluated.Methods Forty-three patients with allopurinol-SCAR,133 patients without SCAR after taking allopurinol for 3 months were included.Ninety-six patients with SCAR caused by other drugs and 148 healthy individuals were enrolled into the present study.HLA-B * 5801 allele was detected by PCR-SSP method.Data were analyzed by chi-square test.Results HLA-B * 5801 was present in 40 of 43 (93.0％) patients with allopurinol-SCAR,which was significantly higher than 19 of 148 (12.8％) in healthy subjects (x2=100.353,P＜0.01,OR=90.5,95％CI 25.5-321.8).But there were no significant differences between allopurinol-tolerant patients and healthy controls(10 of 133,7.5,x2=2.141,P＞0.05,OR=0.6,95％CI 0.2-1.2).And there were only 14 of 96 (7.5％) patients with SCAR caused by other drugs had HLA-B * 5801 (x2=0.152,P＞0.05,OR=1.2,95％CI 0.6-2.4).Conclusion The study indicates that people with HLA-B * 5801 have a high risk of allopurinol-SCAR.HLA-B * 5801 is a specific and predictive marker for guiding the selection of uric acid lowing drug allopurinol.

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.

Aim To study and compare the pharmaco-kinetic parameters of roxithromycin under normoxic and hypoxic rats. Methods A highly effective and rapid ultra-performance liquid chromatography with tandem mass spectrometry ( UPLC-MS/MS) method with posi-tive electrospray ionization source was successfully de-veloped and validated for quantification of roxithromy-cin in rat plasma. Sprague-Dawley rats were randomly divided into the hypoxia and normoxic groups. Each rat obtained a single dose of roxithromycin with 10 mg · kg-1 via intragastric administration. The pharmacoki-netic parameter comparison between normoxic and hy-poxic groups was calculated by SPSS software using in-dependent sample t test method. Results The main pharmacokinetic parameters of roxithromycin between the normoxic and hypoxic rats were:the AUC(0-t) 7 576 and 3 761 μg·h·L-1 , MRT(0-t) 5. 6 and 7. 7 h, T1/2 3. 4 h and 3. 9 h, CL 1. 5 and 3. 0 L · h-1 · kg-2 , tmax3. 1 and 3. 4 h, Cmax 1 116 and 372 μg·L-1 , re-spectively. The levels of Cmax and AUC of roxithromy-cin in hypoxic rats were statistically lower than those in normoxic rats. Conclusion The exposure level of rox-ithromycin in hypoxic rats markedly decreased. Our re-sults may provide an important experimental basis to adjust the dosage for roxithromycin in hypoxic clinical practice.

Objective To establish quantitative method for detection of methylation level of DLC-1 promoter with HRM technology to analyze its association with pathological parameters in prostate cancer.Methods 89 prostate cancer tissue samples and 10 matched normal tissue samples were enrolled into this study.Prostate cancer cells were obtained by LCM.DNA was extracted and modified for methylation determination.CpGenome Universal Methylated DNA was chosen as 100% methylation sample.Then the calibrators representative of 100%,80%,50%,30%,10% and 0% methylation levels were prepared with dilution in a DNA sample of peripheral blood from healthy subjects(100% non-methylation).The DLC-1 methylation levels in prostate cancer tissue samples were detected with HRM.The associations of methylated level with age of patients,PSA value,TNM stage were investigated respectively.ResultsThe melting curves representing 100%,80%,50%,30%,10% and 0% methylation levels were aligned from right to left.The methylation levels of 10 adjacent normal samples and 35 prostate cancer samples were overlapped with 0% methylated calibrator.The methylation levels of 5 cancer samples ranged between 0% and 30%.The methylation levels of 29 cancer samples ranged between 31% and 80%.The methylation levels of 20 cancer samples ranged between 81% and 100%.HRM could be used to reliably detect the as low as 10% methylation for each assay,whereas methylation specific PCR(MSP) could be used to detect 30% methylation level.No significant association between methylation level and patients' age(X~2=3.29,P=0.19),PSA level(X~2=2.04,P=0.36) was found.However,DLC-1 methylation was higher in the prostate cancer tissues with advanced TNM stage(X~2=9.04,P=.01).Conclusions The quantitative method for DLC-1 methylation with HRM is successfully established.It is convenient with good reproducibility and high sensitivity.DLC-1 methylation could be used as the molecular marker for estimation of malignancy in prostate cancer.

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.

Objective By a sequencing panel consisting of 50 targeted genes, aiming at depicting the molecular landscape of ET, PV, and PMF, which are three major subtypes of MPN, to provide valuable information in the diagnosis and prognosis of MPN.Methods A retrospective study was conducted of 53 patients from Huashan hospital and Changhai hospital. All patients were diagnosed in accordance with the 2016 WHO diagnostic criteria for MPN, including 31 cases of ET(11 males, 20 females, median age 55 years), 17 cases of PV(12 males, 5 females, median age 65 years), and 5 cases of PMF(4 males, 1 females, median age 67 years), and underwent next-generation of DNA sequencing of their bone marrow or blood samples. The genetic analyses were performed on bone marrow or peripheral blood. Referring to COSMIC, dbSNP, Clinvar and other public databases, we analyzed the sequencing data, and elucidated the mutation profile of MPN patients, combining with their clinic information. Results In addition to the typical JAK2, CALR, and MPL mutations, pathogenic mutations in other 11 genes were detected, as well as 4 SNPs that confer individual susceptibility to MPNs (rs4858647, rs9376092, rs58270997, rs621940). The average rate of mutated genes was 2.3 genes per patient. In all patients (53 cases), the mutated genes detected were TET2, EZH2, ASXL1, MIR662, SF3B1, BARD1, DNMT3A, KIT, RUNX1, TP53, NRAS according to their mutational frequency. Conclusions Applying next-generation sequencing technology, multi-gene sequencing of a bunch of typical BCR-ABL-negative MPN patients can be performed at one time within 2 working days, and pathogenic mutations other than JAK2, CALR, MPL can be found, which has a bright prospection in clinic.

ObjectiveTo explore the effect of Naoxin'an capsule against mitochondrial dysfunction and oxidative damage in brains of rats with vascular cognitive impairment （VCI） induced by chronic cerebral ischemia. MethodA total of 150 rats were randomized into modeling group （130 rats） and sham-operated group （20 rats） with the random number table method. The two-vessel occlusion （2-VO） was employed to induce VCI in rats， and finally 87 rats developed VCI. The VCI rats were classified into model group， positive drug group （Aricept， 0.5 mg·kg^-1）， and low-， medium- and high-dose Naoxin'an capsule groups （0.18， 0.36， 0.72 g·kg^-1， separately）， with 17-18 rats in each group. The administration lasted 8 weeks. The learning and working memory of VCI rats were assayed by novel object recognition test and Y-maze test. The 8-oxoguanine （8-OxoG） level was measured based on immunofluorescence staining. Spectrophotometry was performed to determine the activity of mitochondrial respiratory chain complexes Ⅰ， Ⅱ， Ⅲ， and Ⅳ， mitochondrial swelling， mitochondrial membrane potential （MMP）， and activity of pyruvate dehydrogenase （PDH） and α-ketoglutarate dehydrogenase （KGDH）. The submicroscopic structure of mitochondria was observed with electron microscope. The phosphorylation of cAMP response element-binding protein （CREB） and the expression of peroxisome proliferator-activated receptor γ coactivator-1α （PGC-1α）， nuclear respiratory factor 1 （NRF-1）， and mitochondrial transcription factor A （mt-TFA） were determined by Western blot. The mitochondrial status Ⅳ H₂O₂ generation， activity of cerebral superoxide dismutase （SOD） and glutathione peroxidase （GSH-Px）， and malondialdehyde （MDA） content were measured with the photochemical method. ResultCompared with the sham-operated group， model group showed decrease in new object discrimination index （P<0.01） and spontaneous alternation rate in the Y-maze test （P<0.01）， increase in ROS production in the brain （P<0.01）， reduction in MMP and mitochondrial swelling A value （P<0.01）， obvious mitochondrial swelling， vacuoles and cristae fractures in mitochondria， decrease in the level of phosphorylated CREB， expression of PGC-1α， NRF-1 and mt-TFA （P<0.01）， and activity of the respiratory chain complexes Ⅰ， Ⅱ， Ⅲ， and Ⅳ （P<0.01）， PDH and KGDH in the brain （P<0.01）， rise of the production of H₂O₂ at state IV （P<0.01） and the content of MDA （P<0.01）， and reduction in the activity of SOD and GSH-Px （P<0.01）. Compared with the model group， each dose of Naoxin'an capsules can improve the new object discrimination index （P<0.05， P<0.01） and the rate of spontaneous alternation （P<0.05， P<0.01）， decrease ROS production in the brain （P<0.01）， improve the MMP and swelling A value （P<0.05， P<0.01）， alleviate mitochondrial swelling and mitochondrial submicroscopic damage， elevate the phosphorylated CREB level， the expression of PGC-1α， NRF-1 and mt-TFA （P<0.05， P<0.01）， and the activity of mitochondrial respiratory chain complexes Ⅲ， and Ⅳ， PDH and KGDH （P<0.01）， decrease state Ⅳ H₂O₂ generation （P<0.01） and MDA content （P<0.05， P<0.01）， and raise the activity of SOD and GSH-Px （P<0.01）. ConclusionBy activating the CREB/PGC-1α pathway， Naoxin'an capsule can protect the structure and function of mitochondria， enhance the antioxidant capacity， and inhibit the mitochondrial dysfunction and oxidative damage induced by chronic cerebral ischemia， thus improving the VCI.

Astrocytes are important nerve cells in the central nervous system （CNS）， which mainly play a key role in nutrition and support. Astrocytes and neurons undergo close energy coupling and substance coupling， which are closely related and interact with each other. In recent years， many studies have shown that the astrocyte-neuron coupling imbalance plays a central role in the occurrence and progression of Alzheimer's disease （AD） and serves as an important therapeutic target receiving increasing attention. According to traditional Chinese medicine （TCM） theory， the main pathogenesis of AD is kidney deficiency and marrow inadequacy， and in clinical medication， kidney-tonifying and marrow-filling TCM prescriptions are often employed with satisfactory results achieved. As reported， many kidney-tonifying and marrow-filling prescriptions exhibit regulatory and protective effects on the imbalance of astrocyte-neuron coupling， suggesting that the effect of kidney-tonifying and marrow-filling prescriptions in treating AD may have some internal relationship with its regulation of the imbalance of astrocyte-neuron coupling. This article reviewed the underlying internal relationship between the imbalance of astrocyte-neuron coupling and the pathogenesis of kidney deficiency and marrow inadequacy in AD and the research progress in the intervention mechanism of TCM for tonifying the kidney and filling the marrow.