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Somatic mutation and novel strategies for personalized targeting therapy
Journal of ZheJiang University(Medical Science), 2013, 42(1): 1-5.   https://doi.org/10.3785/j.issn.1008-9292.2013.01.001
Abstract( 277 )   HTML (   PDF(762KB)( 502 )
Gel immobilization of human genome
Journal of ZheJiang University(Medical Science), 2013, 42(1): 6-13.   https://doi.org/10.3785/j.issn.1008-9292.2013.01.002
Abstract( 240 )   HTML (   PDF(1372KB)( 275 )
Objective: To develop a solid phase PCR method by covalent single point immobilization for recycle utilization of human genome.
Methods: Polymethacrylamide gel was selected as a solid PCR carrier based on DNA-hydrogel copolymer chemistry presented by Mirzabekov.(CH2)6NH2 amino-modified PCR product and randomly fractured formic acid-modified plasmid pGEM-T-HLA-G were used as templates.The specificity of the attachment chemistry was characterized by acrylamide gel electrophoresis,and the thermal stability of method was demonstrated by PCR.This method was applied for the recycle utilization of human genome.Sequencing was used to exclude the possibility of introduced mutations during modification and immobilization procedures.
Results: The PCR detections of plasmid DNA and human genome DNA immobilized by polymethacrylamide gel was successful.The thermal stability of method was successfully demonstrated by performing PCR after 16 rounds of standard 36 PCR cycles.And the sequencing was found no mutation.
Conclusion: The DNA immobilization method with polymethacrylamide gel as a solid phase carrier is stable and specific,which can be a possible approach for realizing recycle utilization of human genome for whole-genome sequencing and SNP detection.
Determination of several environmental contaminants in human body
Journal of ZheJiang University(Medical Science), 2013, 42(1): 14-18.   https://doi.org/10.3785/j.issn.1008-9292.2013.01.003
Abstract( 254 )   HTML (   PDF(634KB)( 306 )
Objective: To detect common environmental pollutants in human body.
Methods: Urine samples were collected from 80 healthy subjects.Chromatography mass spectrometry (GC-MS),HPLC and ELISA were applied to detect several common environmental pollutants in urine samples.
Results: DBP and methylbenzene were present in 75.3% and 41.2% of urine samples.The methanal and AFM1 were found in most of urine samples (approximately 91~97%).By contrast,PCBs,CPZ,4,5-DCC were found in less than 5 samples,but there was no TMT detected.
Conclusion: Some of the environmental pollutants including carcinogens are detected in urine samples in this study.
Preparation of salbutamol polyclonal antibodies and development of indirect competitive enzyme-linked immunoassay
Journal of ZheJiang University(Medical Science), 2013, 42(1): 19-24.   https://doi.org/10.3785/j.issn.1008-9292.2013.01.004
Abstract( 218 )   HTML (   PDF(867KB)( 288 )
Objective: To prepare the antibodies against salbutamol (SAL) with high sensitivity and to develop an indirect competitive enzyme-linked immunoassay (ic-ELISA) for fast detection of SAL.
Methods: The New Zealand white rabbits were immunized with SAL in a small dose and long period mode.The method of ic-ELISA was optimized and adopted for the detection of a series of SAL samples,then the standard curve of SAL was established.The precision and the recoveries of the method were determined.
Results: The antibodies with high sensitivity towards SAL were prepared with a IC50 of 12.21 ng/ml.The ic-ELISA method for SAL measurement was established,the recoveries of measurement was between 95%-105% and the CV was <3%.
Conclusions: The antibodies against salbutamol have been prepared and an indirect competitive enzyme-linked immunoassay for fast and specific detection of SAL has been developed.
Synthesis of artificial diethylstilbestrol antigen for preparation of polyclonal antibodies
Journal of ZheJiang University(Medical Science), 2013, 42(1): 25-31.   https://doi.org/10.3785/j.issn.1008-9292.2013.01.005
Abstract( 207 )   HTML (   PDF(1594KB)( 283 )
Objective: To synthesize artificial diethylstilbestrol (DES) antigen and to prepare DES polyclonal antibody with high titer and sensitivity.
Methods: The derivative of DES (DES-HS) was synthesized from diethylstilbestrol,ethyl bromoacetate,bovine serum albumin (BSA) and chicken ovalbumin (OVA) with the nucleophilic substitution reaction; the compound was identified by electrospray ionization mass spectrometry(ESI-MS).The DES-HS and the carrier proteins (BSA,OVA) were cross-linked to prepare the artificial antigen; the UV absorption spectrophotometry and high performance liquid chromatography (HPLC) were used to identify the prepared artificial antigen.The rabbits were immunized with the DES artificial antigen to prepare the DES polyclonal antibodies.
Results: The DES-HS was synthesized.The DES artificial antigen was prepared successfully with a coupling rate of 22∶1.The DES polyclonal antibodies with a titer of 1∶25 600 and IC50 of 10.81 ng/ml were prepared with DES artificial antigen.
Conclusion: A set of methods to synthesize DES artificial antigen and to prepare the DES polyclonal antibodies has been developed successfully.
Purification of arsenic-binding proteins in hamster plasma after oral administration of arsenite
Journal of ZheJiang University(Medical Science), 2013, 42(1): 32-37.   https://doi.org/10.3785/j.issn.1008-9292.2013.01.006
Abstract( 208 )   HTML (   PDF(1225KB)( 252 )
Objective: To purify the arsenic-binding proteins (As-BP) in hamster plasma after a single oral administration of arsenite (iAsⅢ).
Methods: Arsenite was given to hamsters in a single dose.Three types of HPLC columns,size exclusion,gel filtration and anion exchange columns,combined with an inductively coupled argon plasma mass spectrometer (ICP MS) were used to purify the As-BP in hamster plasma.SDS-PAGE was used to confirm the arsenic-binding proteins at each purification step.
Results: The three-step purification process successfully separated As-BP from other proteins (ie,arsenic unbound proteins) in hamster plasma.The molecular mass of purified As-BP in plasma was approximately 40-50 kD on SDS-PAGE.
Conclusion: The three-step purification method is a simple and fast approach to purify the As-BP in plasma samples.
Optimization of tri-expression of human CYP3A4 with POR and cyt b5 in Sf 9 cells
Journal of ZheJiang University(Medical Science), 2013, 42(1): 38-44.   https://doi.org/10.3785/j.issn.1008-9292.2013.01.007
Abstract( 241 )   HTML (   PDF(1234KB)( 278 )
Objective: To investigate the optimal conditions of tri-expression of CYP3A4,POR and cyt b5 in Sf 9 cells.
Methods: The Sf 9 cells expressing CYP3A4, POR and cyt b5 were cultured in shaker flasks.The optimized conditions,including the temperature and rotation speed,the culture volume,the amount of surfactant and the culture time were studied.The expressed products in microsomes were used to metabolize the testosterone and their metabolic activity was determined.
Results: When the temperature and rotation speed of the shaker were 27℃ and 90 r/min,the cell density and culture volume were 5×105 cells/ml and 80-120 ml per 250 ml shaker flasks,respectively.When Pluronic F-68 was 0.1% and the culture time was 72 h,the condition was most suitable for culture of Sf 9 cells and expression of targeted proteins.When the ratio of the volume of three added viruses was 1∶1∶1,the expression condition was optimal,under which the Km,Vmax,and CLint for testosterone metabolism were 119.6 μmol/L,0.52 μmol/(min·g protein)and 4.34 ml/(min·g protein),respectively.
Conclusions: The conditions of tri-expressing of CYP3A4,POR and cyt b5 have been optimized in the study and the product CYP3A4 is obtained with higher metabolic activity.
AGTR1 A1166C polymorphism is associated with risk of diabetic nephropathy
Journal of ZheJiang University(Medical Science), 2013, 42(1): 45-51.   https://doi.org/10.3785/j.issn.1008-9292.2013.01.008
Abstract( 260 )   HTML (   PDF(1705KB)( 337 )
Objective: To investigate whether the angiotensinⅡtypeⅠreceptor gene (AGTR1) A1166C polymorphism is associated with a high risk of diabetic nephropathy.
Methods: The allele frequency and the genotype distribution of the AGTR1 A1166C polymorphism were studied in normal controls (157 cases),simple diabetes (141 cases,duration of diabetes ≥10 years),and diabetic nephropathy (152 cases) by means of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).
Results: Patients with diabetic nephropathy had a higher frequency of C allele of the AGTR1 A1166C polymorphism than that of normal controls and simple diabetes (P<0.05); but there was no significant difference in frequency of C allele between the normal controls and patients with simple diabetes.
Conclusion: The diabetic patients with AGTR1 C allele may be more susceptible to diabetic nephropathy than diabetic patients with A allele.
Expression and distribution of cysteinyl leukotriene receptors CysLT1R and CysLT2R,and GPR17 in brain of Parkinson disease model mice
Journal of ZheJiang University(Medical Science), 2013, 42(1): 52-60.   https://doi.org/10.3785/j.issn.1008-9292.2013.01.009
Abstract( 286 )   HTML (   PDF(7433KB)( 623 )
Objective: To examine the spatiotemporal profiles and localization of CysLT1R,CysLT2R and GPR17 in mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson disease (PD).
Methods: PD model was induced by subcutaneous injection of MPTP (25 mg/kg) for 5 d in adult male C57BL/6 mice.At d10 after MPTP injection,the expression and cellular localization of CysLT1R,CysLT2R and GPR17 in the substantia nigra were detected by immunohistochemistry and immunofluorescence.
Results: CysLT1R,CysLT2R and GPR17 were normally localized in TH-positive dopaminergic neurons and microglia,while CysLT2R was also expressed in astrocytes.In dopaminergic neurons,approximately 91% co-expressed GPR17,77% co-expressed CysLT1R and 52% co-expressed CysLT2R.Compared with the control group,TH-positive cells in the substantia nigra were significantly reduced in PD mice.CysLT1R,CysLT2R and GPR17-positive cells were significantly reduced; and CysLT1R,CysLT2R,GPR17-positive dopaminergic neurons were also significantly reduced in the PD group.In the striatum,both CysLT1R and GPR17 were normally expressed in neurons; whereas CysLT2R was expressed in astrocytes.In PD striatum,CysLT1R and GPR17-positive cells were decreased,but CysLT2R expression was significantly increased which mainly expressed in the proliferating astrocytes.
Conclusion: CysLT1R,CysLT2R and GPR17 may be involved in the MPTP-induced PD damage in mice.
Expression of 5-lipoxygenase in hippocampal CA1 neuronal damage following global cerebral ischemia in rats
Journal of ZheJiang University(Medical Science), 2013, 42(1): 61-66.   https://doi.org/10.3785/j.issn.1008-9292.2013.01.010
Abstract( 199 )   HTML (   PDF(1320KB)( 233 )
Objective: To determine 5-lipoxygenase (5-LOX) expression and the effect of zileuton,a selective 5-LOX inhibitor,on hippocampal neuron injury induced by global cerebral ischemia in rats.
Methods: Global cerebral ischemia was induced by bilateral common carotid artery occlusion combined with hypotension in rats.5-LOX expression was detected by Western blot analyses and 5-LOX localization was visualized by immunohistochemistry and double immunofluorescence methods.The 5-LOX inhibitor zileuton (10,30,50 mg/kg) was orally administered for 3 d after ischemia.
Results: The 5-LOX expression was increased in the ischemic hippocampus on d1-7 (peaked at d3),and 5-LOX protein was primarily localized in neurons and translocated to the nuclei in the hippocampal CA1 region after ischemia.The 5-LOX inhibitor zileuton (30,50 mg/kg) reduced ischemia-induced hippocampal neurons death 3d after ischemia.
Conclusions: 5-LOX is involved in global cerebral ischemic damage in rats,and the 5-LOX inhibitor zileuton has a protective effect on neuronal damage in the rat hippocampus following global cerebral ischemia.
Construction of a lentiviral RNA interference system targeting heparanase based on miR30 and its silencing effect
Journal of ZheJiang University(Medical Science), 2013, 42(1): 67-74.   https://doi.org/10.3785/j.issn.1008-9292.2013.01.011
Abstract( 276 )   HTML (   PDF(4455KB)( 351 )
Objective: To construct a lentiviral RNA interference system targeting heparanase (HPSE) based on miR30 and to test its silencing effect.
Methods: Three heparanase-shRNA structures were designed based miR30.The targeting fragments were obtained by PCR,then inserted into the vector LV PP-GFP to construct the recombinant lentiviral vector LV PP-GFP/miR-HPSE-shRNA,which was identified by PCR and sequencing.The 293T cells were co-transfect with LV PP-GFP/miR-HPSE-shRNA,pHelper 1.0 vector and pHelper 2.0 vector to produce lentiviruses,with which A375 cells were infected.Real-time fluorescence quantitative PCR and Western blot were performed to evaluate the expression of heparanase RNA and protein.
Results: The lentiviral miR30-based RNAi vector targeting heparanase was constructed and confirmed by PCR and sequencing.The results of real-time fluorescence quantitative PCR and Western blot showed that the expression levels of both heparanase mRNA and protein in infected A375 cells were decreased significantly than those in control group.
Conclusions: The lentiviral miR30-based RNAi vector targeting heparanase was been constructed successfully,which can be used for further study on RNAi-mediated oncolytic viruses.
Morphology and cell proliferation evaluation of follicles from cryopreserved human ovarian tissue by vitrification
Journal of ZheJiang University(Medical Science), 2013, 42(1): 75-80.   https://doi.org/10.3785/j.issn.1008-9292.2013.01.012
Abstract( 190 )   HTML (   PDF(1961KB)( 254 )
Objective: To evaluate the morphology and proliferation of follicles from cryopreserved human ovarian tissue by vitrification.
Methods: Ovarian biopsy specimens were taken from 12 patients.The specimens were randomly distributed into fresh group (Group A) and vitrificated group (Group B).Histological examination and ultrastructural observation were performed after cryopreservation.Both were embedded in paraffin block and proliferating cell nuclear antigen (PCNA) was detected by immunohistochemical staining.
Results: The proportions of primordial and primary follicles from Group A and Group B were 86.4%,13.6% and 84.5%,15.5%,respectively (P>0.05).There was no significant difference in proportions of morphologically normal primordial follicles between Group A and Group B (P>0.05); but the proportion of morphologically abnormal primary follicles was significantly higher in Group B than that in Group A (P<0.05).The ultrastructural studies showed that in histologically normal primordial follicles,there was no difference between Group A and Group B,while there were a few abnormalities of primary follicles in Group B.Granulsa cells and oocytes of primordial and primary follicles and stromal cells were positive for PCNA staining both in fresh and cryopreserved ovarian tissues; there were no differences between two groups.
Conclusion: Vitrification is a favorable method in human ovarian cryopreservation.

Effects of spironolactone on type Ⅰ,Ⅲ collagen concentration in myocardium of spontaneous hypertension rats
Journal of ZheJiang University(Medical Science), 2013, 42(1): 81-85.   https://doi.org/10.3785/j.issn.1008-9292.2013.01.013
Abstract( 308 )   HTML (   PDF(1608KB)( 264 )
Objective: To investigate the effects of spironolactone on the concentration of collagen type Ⅰ,Ⅲ in the myocardium of spontaneous hypertension rats (SHR).
Methods: Twenty 8-week male SHR were assigned randomly into spironolactone (SHR-SPIRO,n=10) and control groups (SHR-CON,n=10),sex-age matched Wistar Kyoto rats (WKY group,n=7) were also served as controls.The rats of SHR-SPIRO group were given 20 mg/(kg·d) of spironolactone,the rats of SHR-CON and WKY groups were given the same volume of distilled water.After 16 weeks,the concentration of collagen type I was analyzed with Western blot.The areas of collagen typeⅠand Ⅲ were observed under polarized light microscopy and the ratio of typeⅠ/Ⅲ collegen was calculated through accumulation score.
Results: Compared with WKY group,the concentration of collagen type Ⅰ in SHR-CON group was significantly higher (1.87±0.2 vs 1.21±0.7,P<0.05).After 16 weeks of treatment the concentration of collagen type Ⅰ(1.42±0.05 vs 1.87±0.2,P<0.05) and Ⅰ/Ⅲ ratio in SHR-SPIRO group were significantly reduced (15.64±1.34 vs 20.8±3.04,P<0.05) compared with SHR-CON group; but there were no differences in accumulation area scores of collagen type Ⅲ among three groups(368.3±30.2 vs 481.6±32.4 vs 406.2±45.3,P>0.05).
Conclusions: The deposition of collagen type Ⅰ in myocardium may be involved in myocardial fibrosis of SHR,and spironolactone can decrease the concentration of collagen type I,which may be one of the mechanisms for its therapeutic effects.
Effects of 17β-estrogen on expressions of C-reactive protein and its mRNA of vascular smooth muscle cells in rats
Journal of ZheJiang University(Medical Science), 2013, 42(1): 86-91.   https://doi.org/10.3785/j.issn.1008-9292.2013.01.014
Abstract( 187 )   HTML (   PDF(1005KB)( 231 )
Objective: To investigate the effects of 17β-estrogen on expressions of C-reactive protein (CRP) and its mRNA in vascular smooth muscle cells(VSMCs).
Methods: Immunocytochemistry was used to detect CRP level in normal VSMCs.The expressions of C-reactive protein and p-ERK1/2 in Ang-Ⅱ-stimulated VSMCs were evaluated with Western blot.C-reactive protein mRNA was examined with RT-PCR.
Results: 17β-estrogen had no effect on cell morphology and C-reactive protein expression in normal VSMCs; however,C-reactive protein and mRNA,as well as p-ERK1/2 were decreased in Ang-Ⅱ-stimulated VSMCs after 17β-estrogen treatment in a concentration-dependent manner.
Conclusion: 17β-estrogen may inhibit the expression of C-reactive protein and its mRNA in Ang-Ⅱ-stimulated VSMCs via ERK1/2 signal transduction pathway in a concentration-dependent way.
Effect of aldosterone on mesenteric resistance vessels in normal or heart failure rats and its mechanism
Journal of ZheJiang University(Medical Science), 2013, 42(1): 92-97.   https://doi.org/10.3785/j.issn.1008-9292.2013.01.015
Abstract( 202 )   HTML (   PDF(1402KB)( 265 )
Objective: To investigate the acute effects of aldosterone (ALD) on mesenteric resistance vessels in normal or heart failure (HF) rats and its mechanism.
Methods: HF model was adopted by in vivo ligation of left anterior descending coronary artery in SD rats; segments of third-order branches of mesenteric artery were isolated and dissected into about 2 mm rings for isometric force recording.
Results: Pretreated with ALD for 10 min,phenylephrine (PE)-induced contraction of normal mesenteric artery decreased first and then increased compared to control group along with the increase of the concentration of PE while decreased in HF rats.This effect was attenuated by ALD receptor-special antagonist eplerenone partially.ALD increased Ach-induced endothelial-dependent vascular relaxation significantly compared to control group both in normal and HF rats.Pretreated with ALD and dexamethasone (DEX) for 10 min,the effects of ALD on PE-induced contraction were weakened in mesenteric artery both of normal and HF rats.And this reaction of DEX to ALD-treated mesenteric in normal rats was attenuated by RU486 partially.
Conclusions: ALD has biphasic effect in PE-induced response on mesenteric artery of normal rats,while reduces the sensitivity of mesenteric artery to PE in HF rats.DEX attenuates the biphasic effect of ALD on artery of normal rat partially but has no significant effect on that of HF rats.
Determination of 7-ethyl-10-hydroxycamptothecin in microdialysates from rat brain with LC-MS/MS
Journal of ZheJiang University(Medical Science), 2013, 42(1): 98-102.   https://doi.org/10.3785/j.issn.1008-9292.2013.01.016
Abstract( 266 )   HTML (   PDF(860KB)( 266 )
Objective: To establish a method for determination of 7-ethyl-10-hydroxycamptothecin (SN-38) in microdialysates from rat brain.
Methods: The concentrations of SN-38 were measured by LC-MS/MS method with Agilent Eclipse Plus C18(2.1 mm×100 mm,1.8 μm)reversed phase column using acetonitrile-0.1% methanoic acid as mobile phase with gradient elution at a flow rate of 0.3 ml/min and temperature at 35℃.Multiple reaction monitoring using the precursor to product ion combinations of m/z 393.1→349.1 was performed to detect SN-38 in microdialysates from rat brain.
Results: Blank microdialysate had non-interference.The method was linear over the concentration range of 0.1015-1015 ng/ml (r=0.9995); and the lower limit of quantification (LOQ) was 0.1015 ng/ml.The recovery of assay for SN-38 ranged from 97.54%-100.60%.The intra- and inter-day precision and stability were both well.The concentrations of SN-38 in brain microdialysates presented pharmacokinetics process and achieved the peak after 220 min.
Conclusions: The fully validated LC-MS/MS analytical method has high specificity and sensibility,which can be used effectively to analyze SN-38 in microdialysates from rat brain.
Whole-genome sequencing: a new approach for understanding of pathogenesis and individualized treatment of cancer
Journal of ZheJiang University(Medical Science), 2013, 42(1): 103-108.   https://doi.org/10.3785/j.issn.1008-9292.2013.01.017
Abstract( 208 )   HTML (   PDF(666KB)( 259 )
With the development of sequencing technology,the cost of whole-genome sequencing was significantly declined.Meanwhile,with the application of combined whole-genome sequencing with epigenetic analysis on methylation and histone acetylation,the comprehensive and systematic analysis of numerous samples became a reality and we are able to re-understand the genesis and development of cancer.New ideas are emerging in comparative genomics research methods,from comparison of genomes among different individuals to horizontal self-comparison of different tissues and vertical self-comparison of genomes recently.Individualized diagnosis and treatment of cancer has shown a bright future.
Heterogeneous expression of human CYP and its application in early stage of new drug research and development
Journal of ZheJiang University(Medical Science), 2013, 42(1): 109-113.   https://doi.org/10.3785/j.issn.1008-9292.2013.01.018
Abstract( 171 )   HTML (   PDF(450KB)( 285 )
Human cytochrome P450 (CYP) has a pivotal role on metabolism of xenobiotics and endogenous substances in clinical practice.Since the CYP from human tissue is very complex,and the human tissue itself is not easy to obtain,investigators begin to use all kinds of expression system to heterogeneously express the CYP.The single CYP expressed was then used for drug metabolism and drug-drug interaction research,to improve the efficiency of high-throughput drug screening greatly.Besides,since the polymorphism of drug-metabolizing enzymes makes efficacy variance for some drugs in different population,the heterougeneous expression and drug metabolizing research of certain CYP mutants will be helpful to guide the optimization of therapeutic regimen and conduct the personalized medication.
AQP4 regulation for cerebral edema
Journal of ZheJiang University(Medical Science), 2013, 42(1): 114-124.   https://doi.org/10.3785/j.issn.1008-9292.2013.01.019
Abstract( 254 )   HTML (   PDF(1069KB)( 410 )
Water balance is one of the basic regulation mechanisms of homeostasis.There are 13 subtypes of aquaporins in mammals (AQP0-AQP12).In neural system,the AQP4 is mainly distributed in astrocytes.Phosphorylation and expression regulation of AQP4 is involved in the formation of brain edema,particularly in the clearance of vasogenic edema and the formation of cytotoxic edema.This article reviews regulations and functions of AQP4 in vasogenic edema and cytotoxic edema.
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