This paper outlines some molecular marking technology based on rDNA sequences and several DNA fingerprinting technology (RAPD, ARDRA, AFLP, REP/ERIC-PCR) used in classification, identifica-tion and diversity research in lactic acid bacteria. The principles, methods and progress in recent years of these technologies were also introduced. At the same time, this paper also compares the advantages and dis-advantages of these methods. People should choose suitable method according to their purposes.

microRNA (miRNA) is a class of endogenous non-coding small RNAs,they play an important role in post-transcriptional regulation of gene expression through combining the target mRNA that the target protein would not synthesis.The present studies have found that miRNA is involved in many kinds of physiological processes,as well as the pathological processes.The abnormal expression of miRNA in many diseases can be used in diagnosis,prognosis and treatment monitoring.But all of these studies depend on an ideal detection method of miRNA.A lot of detection methods of miRNA expression developed from qualitative analysis to quantitative analysis step by step and from single miRNA detection to high throughput screening in recent years.Various detection methods are improved constantly,the specificity and sensitivity has been improved at the same time,detection procedure become simple and practicable,detection time shorten considerably and cost also reduced ceaselessly,which make the application of miRNA in clinical possible.This review highlights the latest research progress of the common detection methods of miRNA.

Objective To investigate the dynamic changes of splenocyte cytokines in mice immunized with the transgenic alfalfa containing Eg95-EgA31 fusion gene of Echinococcus granulosus (Eg). Methods Eighty-eight Balb/c mice were divided into 2 groups randomly according to body weights,and immunized orally or intranasally with 100μl or 10μl extracted leaf protein from the transgenic alfalfa(20 g/L) respectively once per 3 days for 2 months. Four mice randomized from each group were killed to get splenocyte on week 0(control),2,4,6,8,10,12,14,16,18 and 20 after the last immunization. The splenocyte were cultured in medium for 48 hours with EgAg or concanavalin A (ConA) stimulation to induce the interleukin (IL)-12,interferon γ(IFN-γ) and IL-10,and cultured for 72 hours with EgAg or lipopolysaccharide (LPS) stimulus to induce the tumor necrosis factor α (TNF-α). Then the supernatant was collected to measure the level of IL-12,IFN-γ,TNF-α and IL-10 by ELISA. Results In the oral immunization group,the level of IL-12,IFN-γ,TNF-α and IL-10 increased significantly from week 4 to week 6,week 2 to week 8,week 2 to week 6 and week 4 to week 12,respectively,reaching the highest level(25.0±5.8)ng/L on week 4,(575.0±28.9)ng/L on week 2,(50.0±11.5)ng/L on week 2 and (42.5±2.9)ng/L on week 8,respectively,as compared with the values on week 0[(11.3±2.5),(125.0±28.9),(11.3±2.5),(12.5±2.9)ng/L,all P < 0.01]; in the intranasal immunization group,it was similar about the values of IL-12,IFN-γ,TNF-α and IL-10 could be seen from week 4 to week 6,week 2 to week 10,week 4 to week 10 and week 6 to week 16,respectively,reaching the highest level(25.0±5.8)ng/L on week 6,(725.0±28.9)ng/L on week 4,(27.5±2.9)ng/L on week 6 and (60.0±11.5)ng/L on week 6,respectively,as compared with the values on week 0[(11.3±2.5),(125.0±28.9),(11.3±2.5),(12.5±2.9)ng/L,all P < 0.01]. The cytokine levels in the groups with EgAg,ConA or LPS stimulus were significantly higher than those in the corresponding splenocyte suspension groups(P < 0.05 or < 0.01),and the cytokine levels in the groups with ConA or LPS stimulus were obviously higher than those in the corresponding groups with EgAg stimulation (P < 0.05 or < 0.01). Conclusion The mixed responses of Th1 and Th2 types can be induced in mice immunized with the transgenic alfalfa in the early period post immunization(2-10 weeks).

Objective To investigate the change of splenocyte subsets in Balb/c mice immunized with transgenic alfalfa(Medicago sativa)containing Eg95-EgA31 fusion gene of Echinococcus granulosus(Eg) and challenged with Eg protoscoleces.Methods Leaf protein was extracted from transgenic alfalfa containing Eg95-EgA31 fusion gene by heat-coagulation method,and concentration of 20 g/L was used in the study.Meanwhile,leaf protein extracted from the transgenic alfalfa containing blank vector(pBI121)and the normal alfalfa was served as control.Thirty-two female Balb/c mice were randomly divided into 4 groups,8 mice in each group.Oral group was immunized with the leaf protein containing Eg95-EgA31 fusion antigen intragastrically(100μl per mouse);intranasal group was immunized with the leaf protein containing Eg95-EgA31 fusion antigen intranasally(10 μl per mouse);blank vector group was vaccinated intranasally with 10μl leaf protein with blank vector(pBI121);and normal control group was given 100μl normal leaf protein intragastrically.All mice in the above mentioned groups were immunized every 3 days for 2 months.Then,the mice were challenged intraperitoneally with Eg protoscoleces(50 protoscoleces per mouse)8 weeks after last vaccination and sacrified 24 weeks pest infection to separate the splenocytes.Flow cytometry was used to measure the percentages of CD4+ and CD8+ T ceils subsets.Resuits Compared with the normal control group(0.166±0.018,0.083±0.006,2.019 ±0.369),the percentages of CD4+(0.286±0.009)and CD8+(0.102±0.004)T cell subsets and the ratio of CD4+/CD8+(2.814±0.014)in oral group increased significantly (P<0.01 or<0.05).The percentage of CD4+ subset(0.269±0.016)and the ratio of CD4+/CD8+(2.955±0.986) in intranasal group was significantly higher than that ofthe normal control group(all P<0.01).The percentage of CD4+ subset in oral group was significantly higher than that of the intranasal group(P<0.05).No significant difference was found in the percentages of CD4+ and CD8+ T cell subsets and the ratio of CD4+/CD8+ between the blank vector group(0.169±0.018,0.093±0.019,1.852±0.188)and the normal control group(all P>0.05).Conclusions CD4+ T cell may play an important role in the protection induced by transgenic alfalfa vaccine against the challenge of Eg protoscoleces.Intragastrical immunization may be a good route.

Objective To investigate the neuronal nitric oxide synthase (nNOS) distribution in intramural striated sphincter of female rats. Methods 4 female Wistar rats were anesthetized with chloral hydrate. The whole bladder and urethra specimen were harvested, fixed in formalin and imbedded in paraffin wax. Longitudinal and transverse sections were developed with 2 urethra respectively. Routine HE staining and Mallory phosphotungstic acid-hematoxylin staining were used for observation of urethral microstructure. nNOS distribution in intramural striated sphincter was determined with immunohistochemical staining. Results The urethras of female Wistar rat were tubular structure about 1.5～2.0 cm long. From outside inward,the structure included circular striated muscle layer,circular smooth muscle layer,longitudinal smooth muscle layer,dense connective tissue and epithelium. The longitudinal sections showed intramural striated muscle predominates the middle and distal third,not the whole urethral length. The transverse sections showed intramural striated muscle is closed ring shape,about 5～10 layers uneven distributed with more layers in the posterior wall than in the front wall. Strong positive expression of nNOS immunoreactivity was found in some intramural striated muscle fiber. Conclusion nNOS expresses in some intramural striated muscle fiber, surggesting nitric oxide may play an important role on intramural striated sphincter.

Objective To study the effects of nitroglycerin on leak point pressure of chronic complete spinal cord injury female rats. Methods Models of chronic complete spinal cord injury in female rats were established. Changes of leak point pressure were investigated following nitroglycerin 3 mg intraperitoneal injection. Results Leak point pressure decreased from (32.27±15.00) cmH2O to (23.29±9.46) cmH2O. Conclusion There was obvious descent of leak point pressure of chronic complete spinal cord injury female rats following nitroglycerin intraperitoneal injection.

Objective To study the effects of nitroglycerin on urethral sphincter of chronic complete spinal cord injury female rats in vitro. Methods 30 SD female rats with 3 months old were transected at T10 spinal cord. After 8 weeks, the 10 rats with BBB scores ≤5 were selected as experimental group, and other 10 normal rats as control group. The difference of tension in both proximal segment (urethral smooth muscle strips) and distal segment (external urethral sphincter muscle strips) were surveyed before and after nitroglycerin 0.5 mg administration in bath. Results The tension of distal segment in control group reduced from (640.50±91.79) mg to (597.50±92.61) mg, and from (485.50±68.94) mg to (459.90±69.51) mg in experimental group. The difference between proximal segment and distal segment were (222.30±12.14) mg and (42.00±7.86) mg before and after administration in control group, and were (223.00±15.78) mg and (25.60±5.17) mg in experimental group. Conclusion The administration of nitroglycerin 0.5 mg in bath can reduce the tension of both urethral smooth muscle and external urethral sphincter in both normal and spinal cord injured rats. The relaxative effect on external urethral sphincter muscle of normal rats exceed that of injured ones, but approximate on urethral smooth muscle.

Objective To discuss the effect of H2 on osteoclast differentiation of RAW264.7 cells induced by RANKL and RANKL/TNF-α.MethodsRAW264.7 cells were treated with H2 in the presence of RANKL and RANKL/TNF-α.RAW264.7 cells viability was assessed by CCK-8.Test the Oxidative Stress of the induced RAW264.7.The number of TRAP-positive cells were counted under light microscopy.The levels of cathepsin K (CTK) and matrix metalloprotein-9(MMP-9) mRNA were analyzed by real-time PCR.ResultsH2 can not influence the RAW264.7 cell viability but can lower oxidative stress.The significant difference(P<0.05) indicated that H2 could significantly decrease the number of TRAP-positive MNCs.The significant difference among the 4 groups in CTK and MMP-9 genes (P<0.05) indicated that H2 could down-regulate their mRNA expression.ConclusionH2 can reduce the oxidative stress and inhibit differentiation of RAW264.7 cells into osteoclasts.