Objective To develop and optimize the extraction method of exosomal miRNA and compare it with traditional methods. Methods The exosomal miRNA of MSC,NK and CIK cells was used as the research subject. The removal efficiency of genomic DNA from exosomal miRNA by gDNA removal column was detected by UV spectrophotometer and agarose gel electrophoresis. The lysates(exosome G2 lysate and exosome miRNA lysate)and aggregation reagents(absolute ethanol and isopropanol)were optimized by using concentration,purity and gene expression level(Ct value)as evaluation indexes.Exosomal miRNA of MSC,NK,CIK cells and healthy human serum was extracted by the developed method,Trizol method and Trizol magnetic beads method,and detected by RT-qPCR. Results The gDNA removal column effectively removed genomic DNA from exosomal miRNA. The concentrations of exosomal miRNA extracted from MSC,NK and CIK cells by using exosome miRNA lysate were significantly higher than those by using exosome G2 lysate(t = 6. 358,P = 0. 020). The purity of miRNA samples extracted by exosome G2 lysate was low,and there was foreign protein pollution,but exosome miRNA lysate effectively removed the pollution. The Ct values of miR-Let-7i,miR-16-1 and miR-150 genes in exosomal miRNA of CIK cells extracted by exosome miRNA lysate were significantly lower than those by exosome G2 lysate(t = 30. 120,P =0. 008). The concentration of exosomal miRNA extracted from MSC,NK and CIK cells by isopropanol was significantly higher than that by absolute ethanol(t = 8. 567,P = 0. 010),and the purity was significantly lower than that by absolute ethanol(t = 6. 214,P = 0. 020). The Ct values of miR-Let-7i,miR-16-1 and miR-150 genes in exosomal miRNA extracted from CIK cells by two aggregation reagents had no significant difference(t = 2. 297,P = 0. 120). Compared with Trizol method and Trizol magnetic beads method,the expression of miR-Let-7i,miR-16-1,miR-Let-7a and miR-150 genes in exosomal miRNA extracted from CIK,NK,MSC cells and healthy human serum by the developed method was more abundant,and the overall Ct value was lower. The dissolution peak was prominent and sharp,exhibiting a single main peak. Conclusion The exosomal miRNA extracted by the developed method has high concentration and purity with stable Ct value,and the method has high sensitivity and good specificity. This study lays a foundation for further research on exosomal miRNA.

[Objective]To study the curative effect and superiority of one stage anterior-posterior approach total spondylectomy and spinal reconstraction in the treatment of thoracolumbar malignant neoplasms.[Method]Twenty-four thoracolumbar malignant neoplasms patients treated by anterior approach total spondylectomy,spinal reconstractin with titanium mesh cage associated with bone graft or bone cement and posterior approach transpediuclar screw fixation.[Result]The follow-up of postoperation was from 9 to 35 months.All patients achieved relieve of pain after surgery,9 cases with incomplete paraplegia improved by 1.8 grades in average with Frankel neurological classifications,2 cases with dysfunction of urination and defecation were recovered,4 patients decease for metastasis in critical organ,all the patients showed bony fusion at 6~9 months,no internal fixation loosening or breakage case was found,1 case was recrudescent after one year.[Conclusion]Treatment of thoracolnmbar malignant neoplasms by one stage anterior-posterior approach total spondylectomy and spinal reconstraction can remove the neoplasms efficiently,reconstraction the spinal stabilization and improve the quality of life.

Objective To introduce a proposal that can standardize the clinical information of urine manifestation in Chinese medicine.Methods A hypothesis of symptomatic units was proposed in this paper,herein,the attribute,name and definition of the symptomatic units of urine manifestations in the clinical records of Chinese medicine in the past dynasties were summarized according to the stipulation of logic classification and denomination of a scientific term.Results The 23 symptomatic units,2 bounds and their assembly can express 3867 descriptions on the clinical manifestation of urine.Conclusion The symptomatic unit is a feasible hypothesis that can simplify the clinical information of Chinese medicine.The attribute extraction,denomination and definition may become one of the effective procedures to standardize the clinical information of Chinese medicine.

Objective Determine the common syndrome elements of viral hepatitis.Methods Refering to the syndrome elements hypothesis proposed by Zhang Zhibin,syndrome elements from the collection of 19,341 cases of Viral Hepatitis were extracted which were diagnosed by National Science and Technology Major Project of AIDS and Viral Hepatitis and Other Major Infectious Diseases Prevention and Control.And the results were compared with industry standards.Results There were 27 involved syndrome elements of viral hepatitis.Common syndrome elements were Qi stagnation,internal dampness,internal fire,blood stasis,yin deficiency,Qi deficiency,toxin,pathogenic water,and yang deficiency; The main excessive syndromes were Qi stagnation,internal dampness,internal fire,and blood stasis.The main deficiency syndromes were yin deficiency and Qi deficiency.Conclusion Excessive syndromes were the main syndromes of viral hepatitis.Dampness,heat,blood stasis,and toxin played key roles in the development of viral hepatitis.In addition to this,Qi stagnation,pathogenic water and phlegm were also important syndrome elements.

Objective:To standardize the clinical information of the nose.Methods:Based on the 1034 descriptions on the clinical manifestation of nose in the database of the clinical records of Chinese medicine in the past dynasties,according to the hypothesis of symptomatic unit,the symptomatic units,the second-level clinical characteristics and the limiting factors were summarized in the paper.Results:All the descriptions on the clinical manifestation of nose include 11 symptomatic units,7 second-level clinical characteristics and 1 limiting factor.Conclusion:Comparing with the symptoms in the authoritative works of Chinese medicine,cold-feeling of the nose while breathing was the new fi nding,nares flaring and ozena recorded in the authoritative works were the second-level clinical characteristics of polypnea and rhinorrhoea respectively.Nose diseases,such as nasal eczema,were usually diagnosed by several symptomatic units,so they were not symptomatic units.

Objective To Analyze CD4+ cell count which embodies curative effect in HIV and patients with AIDS, who have treated with TCM for half a year. Methods According to CD4+cell count, the patients were divided into 4 phases. Their CD4+cell count were analyzed before and after the treatment. Results 1.Rank sun test showed that CD4+cell count were significantly improved in people who used TCM treatment. On the whole, CD4+cell count was(317.76±175.61) in 1 cu mm before treatment, which was(350.60±175.92) in 1 cu mm after treatment, P<0.01. 2. Ridit showed that patients whose CD4+cell count more than 500 were in phase I. Their R=0.614, and the 95%confidence interval was 0.5702 to 0.6579. Patients whose CD4+cell count more than 350 and less than 500 were in phase II. Their R=0.575, and the 95%confidence interval was 0.5439 to 0.6062. Patients whose CD4+cell count more than 200 and less than 350 were inphase III. Their R=0.460 and the 95%confidence interval was 0.4347 to 0.4849. Patients whose CD4+cell count less than 200 were in phase IV. Their R=0.428, and the 95%confidence interval was 0.3971 to 0.4589. There was no overlap between phase III, phase IV and phase I, phase II in 95% confidence interval. Conclusion TCM has the advantages in strengthening vital qi, but that is worse than western medicine in effort of expelling pathogen.

AIM: To study the role of TGF-?/Smad pathway in the development of renal fibrosis in diabetic nephropathy.METHODS: Rats were induced to diabetic nephropathy by using tail intravenous injection of STZ.The expression of TGF-?_1,Smad2/3 protein and mRNA in kidney were examined at 2,4,8 and 16 weeks after STZ induction.CTGF,collagen-Ⅲ,PAI-1 mRNA expression in kidney at 16 weeks of STZ-induced diabetic nephropathy and normal rats were studied by RT-PCR.RESULTS: Weak TGF-?_1,Smad2/3 protein were detected in normal renal tissues while strong TGF-?_1,Smad2/3 staining were observed in renal tissues of diabetic nephropathy(0.057?0.030/0.223?0.040;0.017?0.010/0.153?0.010,respectively,P

Objective This study was performed to investigate the levels of HSP70 in tissue in pemphigus as a possible new theoretical basis for further elucidate the pathogenesis of pemphigus.Methods The expression of HSP70 in 62 patients with pemphigus was determined by immunohistochemistry,and the normal skin was taken as control.Results The results showed that the positive cells of HSP70＞75 ％ in the blisters of pemphigus vulgaris and the positive cells of HSP70＞50％ in the inflammatory cells near the blisters,and the expression of HSP70 was significantly higher than that in normal skin,which was statistically significant(Z=5.42,4.73,P＜0.01).Conclusion The abnormal expression of HSP70 in inflammatory cells and psoriasis of pemphigus patients showed that HSP70 is involved in the pemphigus.

Objective To assess the long-term survival and prognosfie factors in a series of patients with distal bile duet carcinoma. Methods A retrospective clinical analysis was made on 76 cases of distal bile duct cancer who were admitted into our hospital from January 1996 to December 2006. Clinicopathologic factors with possible prognostic significanees were selected and analyzed. Survival was calculated with the Kaplan-Meier method. A multivariate analysis of these individuals was performed using the Cox proportional Hazards Model. Results There were 46 males and 30 females. The age ranged from 21 to 88 years with a mean of 65.21 patients received palliative surgery including, bypass procedure, intraoperative biliary stenting, or percutaneous transhepatie biliary drainage. Radical resection was performed on 42 cases and the 1-, 3- and 5-yeur survival rates were 88.0%, 41.3% and 29.2% respectively. 38 cases died of liver metastasis or recurrence. In multivariate analysis, surgical procedure (P = 0.006) and liver metastasis (P = 0.008), but not sex, age, invasion of pancreas, invasion of duodenum, diameter or differentiation of tumor, were significant independent prognostic factors. Conclusions Radical resection is only curative treatment modality. Prevention on postoperative liver metastasis is essential for improving survival.

AIM: To prepare the vaccine of DCs(pcDNA3-hCEA) and observ the immunity effect of the DCs(pcDNA3-hCEA) inoculating on CT26(hCEA +) loaded in BALB/c mice. METHODS: DCs were generated from bone marrow in the presence of rmGM-CSF and rmIL-4. A new recombinant plasmid, pcDNA3-hCEA, reformed with inserting a 2.4 kb human CEA cDNA into pcDNA3. DC vaccine was prepared by transfection with pcDNA3-hCEA using lipofectamine. CEA mRNA expressed in DCs(pcDNA3-hCEA) was confirmed by RT-PCR, CEA expression level was detected with RIA method, and CEA specific CTL was induced in vitro . After vaccination of DCs(pcDNA3-hCEA), the survival time of the BALB/c mice challenged with critical loading CT26(hCEA +) was observed. RESULTS: G418 test showed that about 14% DCs were transfected with pcDNA3-hCEA. And CEA mRNA and protein could be detected respectively by RT-PCR and RIA in the genetically modified DCs. Furthermore, the DCs coud be targeted by specific CTL, the survival time of the mice challenged with CT26(hCEA +) was prolonged 1-4 weeks. CONCLUSION: These results demonstrate that specific antitumor immune responses could be induced efficiently by vaccination of DCs(pcDNA3-hCEA), which is transfected eukaryotic expression vector encoding tumor antigen gene.