Objective:To clone human PRKCB1 gene open reading frame(ORF) in order to further research on it's function.Methods:RT-nested PCR method was adopted to the amplify the total length of human PRKCB1 ORF from human umbilical vein endothelial cell(HUVEC).T vector was inserted into the harvested fragment after a tail was added.With blue white screening,the gene ORF encoding human PKC?Ⅱ was obtained by special primers amplifying recombinant plasmids,and linked into T vector.Then the plasmid was identified by sequencing.Results:The human PRKCB1 ORF was amplified successfully with RT-nested PCR and T/A cloning,and the gene sequence was completely consistent with that reported in GenBank.Conclusion:Human PRKCB1 gene ORF was successfully cloned.The strategy of cloning may provide technical references for some genes hard to be cloned.

Objective: To observe the influencing factors for efficiency of liposome-mediated transfection into human umbilical vein endothelial cell(HUVEC). Methods: Transferring HUVECs by distinct conditions,such as ratios of liposome and plasmid,densities of per well and different times of incubation,and transfection rates were observed and calculated by fluorescent microscope and flow cytometry. Results:⑴ When density of per well exceeded 2?l04 and times exceeded 4 hours,liposome-mediated transfection efficiency of endothelial cells decreased. ⑵With adding plasmid quality, transfection rates increased;while plasmid quality exceeded 1.0 ?g,in condition of 2?l04 per well and liposome volume 2 ?l, transfection efficiency reached the peak level. ⑶With adding liposome volume, transfection rates increased;while liposome volume was 8 ?l,in condition of 2?104 per well and plasmid quality 1 ?g, transfection efficiency was depressed.When ratios of liposome and plasmid were 1:6～1:8,the optimal transfection efficiency was 18.62%. Conclusion: Using optimal transfection parameter,we obtained the optimal transfection efficiency .

Objective Toinvestigatetheclinicalfeaturesandsurgicalprognosisofmoyamoya syndromeinchildren.Methods Theclinicaldataof12childrenwithmoyamoyasyndromeadmittedto the 307th Hospital of People′s Liberation Army from December 2002 to October 2013 were analyzed retrospectively. Eleven of them underwent encephalo-duro-arterio-synangiosis (EDAS). A total of 550 children with moyamoya disease in the same period were used as a control group. The clinical characteristics and surgical efficacy of the children with moyamoya syndrome were summarized and concluded by comparing the clinical data of the two groups,including sex,age of onset,initial symptom,progress symptoms, Suzukiinstallments,imagingfeatures,andsurgicalefficacy.Results Themaleandfemaleratioof the children with moyamoya syndrome was 1∶2. Their mean age of onset was 12 ± 5 years old. There were significant differences in the initial symptom (cerebral infarction and cerebral hemorrhage )and disease progress between the children with moyamoya syndrome group and the control group (5/12 vs. 14. 5%[80/550], 3/12 vs. 61. 8%[340/550],and 5/12 vs. 8.7%[48/550],respectively;all P<0. 05). Within the follow-up period,of the 11 children underwent EDAS,7 cases had no further attack,and 4 cases were improved significantly. There was significant difference in the modified Rankin scale (mRS)between the beforeandaftersurgery(0[0,1]vs.2[1,2];P<0.05).Conclusions Theclinicalfeaturesofthe children with moyamoya syndrome have some differences with those with moyamoya disease. Timely and effective EDAS treatment may effectively prevent disease progression and improve the prognosis of patients.

ObjectiveToinvestigatetheclinicalfeatures,riskfactorsforbleedingandtreatment outcomes in moyamoya disease patients w ith intracranial aneurysms. Methods The clinical symptoms, location and size of aneurysm, treatment and the long-term folow-up results of the moyamoya disease patients w ith intracranial aneurysms w ere analyzed retrospectively. Results A total of 34 moyamoya disease patients w ith intracranial aneurysms (35 aneurysms) w ere enrol ed, including 22 (64.7%) in the intracranial hemorrhage group and 12 ( 35.3%) in the non-intracranial hemorrhage group. Of the 35 intracranial aneurysms, 23 (main artery type 11, peripheral artery type 12) w ere in the intracranial hemorrhage group and 12 (main artery type 11, peripheral artery type 1) w ere in the non-intracranial hemorrhage group. There w ere 29 smal aneurysms and 6 medium aneurysms (al w ere patients w ith hemorrhagic moyamoya disease). The aneurysms w ere mainly peripheral arterial type in the intracranial hemorrhage group, and the aneurysms w ere mainly artery type in the non-intracranial hemorrhage group. There w as significant difference in aneurysm typing betw een the tw o groups ( P= 0.013 ). Tw o patients did not perform encephalo-duro-arterio-synangiosis (EDAS) in the intracranial hemorrhage group, other patients and those of the non-intracranial hemorrhage group performed EDAS. Angiographical reexamination revealed that 3 patients w ith peripheral aneurysm disappeared, and 1 aneurysm recurred after aneurysm embolization, and the remaining aneurysms did not have any change. Long-term fol ow-up show ed that 1 patient died of sudden cerebral hemorrhage at 1 year after procedure in the intracranial hemorrhage group, and the others did not have ischemic or hemorrhagic stroke. The modified Rankin scale scores w ere improved in 21 patients. Conclusions There are differences in moyamoya disease patients w ith intracranial aneurysm typing w ith different clinical manifestations. Moyamoya disease patients w ith intracranial aneurysms are mostly smal aneurysms and they can not temporarily be treated directly and can perform EDAS directly. Intracranial aneurysms after procedure may remain long-term stability, and some peripheral aneurysms may disappear.

Objective To study the relation between copper,copper-zinc ratio in blood and blood-fat only in the copper intake lower than the normal acceptable daily intake,and to provide a unified dosage evidence for copper multi-ways exposure.Methods Forty-two SD rats were divided into 7 groups randomly.The rats in one group among them as the normal control group were fed on the normal fodder and water,the other 6 groups were fed on special fodder without copper and deionized water and i.g.copper gluconate at doses of 0ADI(the acceptable daily intake of rat),1/625ADI(0.000 128 mg/ml),1/125ADI(0.000 64 mg/ml),1/25ADI(0.003 2 mg/ml),1/5ADI(0.016 mg/ml) and ADI(0.8 mg/ml),for 30 days.The activity of ceruloplasmin(CP),the content of copper,zinc in the blood and the level of total cholesterol(TC),triglyceride(TG),high-density lipoprotein(HDL) and low density-lipoprotein(LDL) were determined.Results In the case of copper intake lower than the normal acceptable daily intake,the activity of CP in the blood of all treated rats were lower than the normal level(P

Objective To investigate the clinical features of moyamoya disease in children and the prognosis of encephaloduroarteriosynangiosis ( EDAS) . Methods According to the age of first operated patients,317 children with moyamoya disease who received EDAS from January 2004 to December 2010 were divided into 3 groups:infant group (n=16,<3 years of age),preschool group (n=42,3 to 6 years of age),and adolescent group (n=259,6 to 17 years of age). The clinical data and the efficacy of operation of the patients were analyzed retrospectively. Results (1) Among the 3 groups of patients,the incidences of cerebral infarction in the infant group (81. 2%,13/16) or the preschool group (69. 0%,29/42) before procedure were significantly higher than the adolescent group (48. 3%,125/259). There were significant differences (χ2 =11. 741,P<0. 01). (2) Before surgical intervention,the infarct volume enlargement or the recurrence of infarction rate at different parts of brain in the infant group (62. 5%,10/16) was higher than that of the preschool group (31. 0%,13/42) and adolescent group (3. 9%,10/259). There was significant difference (χ2 =77. 437,P <0. 01). (3) The overall rate of favourable prognosis was 86. 4% (274/317). There were significant differences between the 3 groups (χ2 =9. 026,P<0.02). Conclusion The conditions of children with moyamoya disease progresses rapidly and their clinical prognosis is poor. It is safe and effective to perform EDAS early moyamoya disease in children.

OBJECTIVE:To provide a reference for innovative drugs to enter the health insurance directory smoothly and pro-mote the rapid development of pharmaceutical industry in China. METHODS:Based on related policy study of foreign and domes-tic innovative drugs entering the health insurance directory,the policy barriers of innovative drugs entering the health insurance di-rectory were analyzed and the countermeasures were put forward. RESULTS & CONCLUSIONS:In China,the foundation of drug selection was not objective enough;the review results were so vulnerable to subjective views because of the experts selection mech-anism;the health insurance directory deletion mechanism was deficient;the waiting time for innovative drugs entering the health in-surance directory was too long;innovative drugs negotiation mechanism was imperfect,etc. It is recommended that forcibly requir-ing pharmaceutical companies should provid the related data about pharmacoeconomic evaluation;the independence and pluralism of the expert group should be enhanced;drugs in the health insurance directory should be secondarily evaluated regularly;the inno-vative drugs should be given the green channel;innovative drug price negotiation rules should be unified to promote drug informa-tion sharing.

Objective To construct the endothelial cell model with overexpressed human protein kinase C ?2(PKC?2) after high glucose inducement in order to study the function of human PKC?2. Methods The PRKCB1 gene was amplified from pMD18-T-PRKCB1 plasmid and then directly cloned into shuttle plasmid pDC315 to construct shuttle plasmid. Then the recombinant shuttle plasmid and adenovirus genomic plasmid pBHGlox△E1,3Cre were cotransfected into 293 cells to construct recombinant adenovirus Ad-PRKCB1. The virus titer was calculated by TCID50. The Ad-PRKCB1 was verified by immunocytochemistry and RT-PCR. Ad-PRKCB1 was transfected into human umbilical vein endothelial cells (HUVECs) followed by the treatment of high glucose (25 mmol/L) for 96 h, and then the cells were observed by laser scanning confocal microscopy (LSCM). Results Restrictive endonuclease digestion and PCR result showed that the target gene was correctly cloned into shuttle plasmid. Cytopathic effect (CPE) was observed under inverted microscope through homologous recombination. The virus titer was 7.9?109 IU/ml. Immunocytochemical staining and RT-PCR indicated the expression of human PKC?2 in the transfected HUVECs. The translocation was observed under LSCM. Conclusion A recombinant adenovirus vector with human PKC?2 and the endothelial cell model with human PKC?2 overexpression by high glucose are constructed successfully.

Objective To construct the eukaryotic expression vector of human PRKCB1 containing enhanced green fluorescence protein gene and transfer into human umbilical vein endothelial cells(HUVECs),then identify activity of expression protein.Methods The pReceiver-M29-PRKCB1 eukaryotic expression plasmid was constructed by frame amplified from pMD18-T-PRKCB1 plasmid.Then the recombinant plasmids were identified by enzyme analysis and DNA sequencing.According to optimized conditions,the eukaryotic expression plasmids were transfered into HUVECs and observed under fluorescence microscope.After that,transfection efficiency was calculated under random vision.The plasma membrane/cytosol ratio of fluorescence was calculated under confocal microscope.The translocation was identified.Results The gene sequence was completely consistent with that reported in GenBank.The enhanced green fluorescence protein was observed in HUVECs after 48 hours.Transfection efficiency was 18.6%?1.6%.The translocation was observed.Conclusion The eukaryotic expression plasmid is successfullyconstructed and transfered into HUVECs,the translocation was identified.It is a potential tool for screening HUVECs stably expressing human protein kinase C ?2 and isolating protein complex.