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 cultivate and identify the transgenic affalfa containing Echinococcus granulosus Eg95 gene. Methods The alfalfa plants were transformed by co-cultivating alfalfa cotyledons via recombinant Agrobacterium tumefaciens LBA4404 harboring pBI-Eg95. The transgenic alfalfa explants were selected by kanamyein after calli formation, shoots and roots regeneration in the selective medium, the seedlings of transgenic plants were obtained which were finally transplanted into pots containing nutrient soil. After 2-3 months growth, the complete transgenic alfalfa plants containing Echinococcus granulosus Eg95 gene were obtained. To identify the transgenic alfalfa plants, the total DNA, RNA and leaf protein were extracted from fresh leaf tissue of the transgenic alfalfa plants and confirmed by PCR, RT-PCR, SDS-PAGE and Western blot assay. Results A specific band around 471 bp was amplified by PCR with total DNA, and the same band was obtained by RT-PCR with total RNA, which confirmed that the Eg95 gene was stably integrated into the transformed alfalfa genome. SDS-PAGE analysis showed that the relative molecular mass(Mr) of the expressed protein was about 16.5×103, consistent with the Eg95 protein, and the level of Eg95 expression was up to 0.06% of total soluble leaf protein by Bio-Rad Quantity one assay. Western blot verified the expressed protein was reactive with the sera of mice infected with Echinococcus granulosus. Conclusion The transgenic alfalfa plants containing Echinococcus granulosus Eg95 gene are successfully cultivated.

Background Carbachol solution (0.01％) is an agonist of M cholinoceptor and skeletal muscle N cholinoceptor,and it is used to play miotic effect and open peripheral iridectomic hole during the surgery of implantable collamer lens (ICL) implantation in order to lower intraocular pressure (IOP).However,the anterior chamber injection of 0.01％ carbachol solution often causes relevant complications,while whether lower dose of carbachol solution is effective and safe is unclear.Objective This study was to compare the effectiveness and safety between 0.01％ carbachol solution and 0.005％ carbachol solution after anterior chamber injection during the ICL implantation.Methods One hundred and fifty-two eyes of 76 cataract patients were included in Daping Hospital of Third Military Medical University from September 2014 to September 2015.ICL implantation and periphery iridectomy were carried out on both eyes of the patients and the 0.01％ carbachol solution was injected into the anterior chamber during the surgery of the right eyes and 0.01％ carbachol solution was used in the left eyes.The operation duration and IOP at postoperative 2 hours and systemic choline-like reaction were compared between 0.01％ carbachol solution group and 0.005％ carbachol solution group.Results The mean operation duration was (11.86± 2.39) minutes and (11.22 ± 1.85) minutes in the 0.01％ carbachol group and 0.005％ carbachol group,without significant difference between two groups (t =1.851,P =0.066).IOP was (15.76 ± 2.18) mmHg and (13.58 ±2.24)mmHg in the 0.01％ carbachol group before and after surgery,and those in the 0.005％ carbachol group was (15.70±2.35)mmHg and (13.12±2.17)mmHg,there was no significant difference in the IOP between the two concentrations of carbachol (Fsroup =0.986,P=O.322).The IOP at postoperative 2 hours was lower than that before operation,the difference was statistically significant(Ftime =97.339,P =0.000).There was no interaction between drug concentration and time (Fcorrelation =0.772,P =0.381).The incidences of complications,such as dizziness,nausea and vomiting,were lower in the 0.005％ carbachol group than those in the 0.01％ carbachol group (x2 =13.01,5.16,4.03,all at P＜0.05).Conclusions Carbachol solution (0.005％) can play intraoperative miosis effect and maintain effective operation duration in ICL implantation.In addition,the application of 0.005％ carbachol solution is quite safe in both intraoperation and postoperation.

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.

Objective To study the cholesterol nuclear-cytoplasmic interaction effect and position cholesterol traits QTL in mice.Methods Improving the nuclear-cytoplasmic interaction models and methods that have been constructed, and analyzing the public database of total cholesterol and lipoprotein data of F2 group that derived from DBA/2J ( D2) and CAST/EiJ ( CAST) mice.Results Six QTL that controlling total cholesterol, HDL and nonHDL were located in 4 linkage groups in the genome.In the models constructed in this study, we found a QTL has significant interaction with cytoplasmic background, which changes the previous results of data analysis, the genetic mouse cholesterol and lipoprotein components opened up new ideas.Conclusion Mouse cholesterol trait is the result of interaction of nuclear genes and cytoplasmic background.