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.

Objective To investigate the clinical and angiographic features in hemorrhagic moyamoya disease patients with the posterior circulation involvement (PCI). Methods The clinical and imaging data of 224 patients with hemorrhagic moyamoya disease from December 2002 to December 2011 were analyzed retrospectively. The patients were divided into either a PCI group (n=57)or a non-PCI group (n=167) according to whether they had PCI or not. Results (1)Suzuki staging concentrated in stageⅤandⅥin the PCI group,accounting for 42. 1%(24/57)and 40. 4%(23/57)respectively;Suzuki staging concentrated in stage Ⅲ and Ⅳ in the non-PCI group,accounting for 31. 1%(52/167)and 41. 9%(70/167)respectively. There was significant difference in the distribution of Suzuki staging between the patients of both groups (P <0. 01). (2)In the PCI group,the thalamic hemorrhage,intraventricular hemorrhage,ganglia hemorrhage,subarachnoid hemorrhage accounted for 43. 9%(26/57),38. 6%(22/57), 3. 5%(2/57),14. 0%(8/57),respectively;There was no cerebral lobe hemorrhage. In the non-PCI group, the thalamic hemorrhage,intraventricular hemorrhage,basal ganglia hemorrhage,subarachnoid hemorrhage and cerebral lobe hemorrhage accounted for 0,37. 7%(63/167),25. 1%(42/167),15. 6%(26/167),21.6 (36/167),respectively. The proportion of hemorrhage types between the PCI group and non-PCI group was significantly different(P<0. 01). (3)The dilation of anterior,posterior choroidal artery ,and patency of posterior communicating artery,posterior pericallosal artery in bleeding side accounting for 19. 3%(11/57),82. 5%(47/57),36. 8%(21/57),78. 9%(45/57)in PCI group and 36. 5%(61/167),3. 0%(5/167),68. 9%(115/167),80. 2%(134/167)in non-PCI group. The ratio of of abnormal vessels between PCI group and non-PCI group had significantly statistical difference (P<0. 01). Conclusion Hemorrhagic moyamoya with PCI is common. Suzuki staging is usually in the advanced stage of vascular staging of moyamoya disease. The thalamic hemorrhage is the main type of bleeding. Posterior choroidal artery rupture may be the main reason of hemorrhage.

MicroRNA(miRNA) is involved in virtually all biologic processes, including cellular proliferation, apoptosis, and differentiation. Thus, miRNA deregulation often results in impaired cellular function and disease development, so miRNAs have potential therapeutic relevance. The elucidation of these processes regulated by miRNAs and the identification of novel miRNA targets in the pathogenesis of hypertension is a highly valuable and exciting strategy that may eventually led to the development of novel treatment approaches for hypertension. Several mechanisms have been implicated in the pathogenesis of hypertension: overactivation of therenin-angiotensin-aldosterone system (RAAS), dysfunction of the vascular endothelium, damnification of vascular smooth muscle. To maintain and restore target organ expression of miRNA stable may be a new strategy for treatment of hypertension. The article reviews pathogenesis of miRNA and hypertension, researches of miRNAs as biomarker and therapeutic target, discusses advances in miRNA-based approaches that may be important in treating hypertension.
Animals ; Biomarkers ; metabolism ; Humans ; Hypertension ; drug therapy ; genetics ; metabolism ; MicroRNAs ; genetics ; metabolism ; Molecular Targeted Therapy

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 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 study glucose,creatinine,urea nitrogen and serum ET-1 of patients with acute cerebral infarction,and to explore the relationship between neurologic impairment and ET-1 levels.Methods The glucose,creatinine,urea nitrogen and serum ET-1 were retrospectively analyzed in 50 patients with acute cerebral infarction ( ＜ 24 h) and 50 patients with non-neurological diseases.ET-1 determined by 125I radioimmunoassay.Results There were no significant differences in glucose,creatinine and urea nitrogen of acute cerebral infarction ( P ＞ 0.05 ) ; Compared to the control groups,ET-1 levels was significantly higher ( P ＜ 0.01 ),and levels of serum ET-1 in acute cerebral infarction were significantly correlated with their neurological deficits ( P ＜ 0.01 ).Conclusions Levels of serum ET-1 can severd as diagnostic and prognostic indicator of acute cerebral infarction.

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.