The ScFv gene containing Nco I and Bam HI was amplified by PCR, and inserted into the prokaryotic expression vector pHOG21 with Nco I and Bam HI digestion. After screening, high-level expression recombinant XL1-Blue (pHOG2E3) were obtained, and the different expression levels of ScFv were detected by SDS-PAGE and EUSA. When the strain was induced by 0.5 mmol/LIPTG and 0.4mol/L sucrose in the culture medium LB at 37℃ for 6 hours, the high level production of ScFv protein was obtained. The molecular weight of the expressed ScFv is 31, 000 D. The ScFv was mainly in the form of inclusion body, but it could also be in the culture medium and soluble periplasmic content. Above all, the ScFv protein could neutralize the phospholipase C activities of alpha-toxin of Gostridium perfrigens type A.

Background The circulating endothelial progenitor cells (EPCs)play an important role in postnatal vasculogenesis and restoration of endothelial injury.Previous investigation illustrated that a reduced ocular blood flow and vascular dysfunction caused by endothelial dysfunction plays a role in the pathogenesis of glaucoma.However,endothelial system accommodation is accomplished with the circulating EPCs.Objective The present trail was to investigate EPCs change in patients with primary acute angle-closure glaucoma (PACG)and explore the role of EPCs in the pathogenesis of PACG. MethodsA prospective cohort study was designed.Thirty patients with PACG were enrolled in Tianjin Medical University General Hospital as PACG group,and 20 normal subjects served as control group.Periphery bloodsamples were obtained fromall the patients and thenstained with saturating concentrations of monoclonal antibodies,FITC-conjugated anti-CD34 and CD133 mAb.EPCs identified by CD34,CD133 were enumerated by flow cytometry,and the correlation between EPCs change and its relative factors was analyzed.Informed consent was obtained from each subject before any medical procedure. Results No significant differences were found in age,gender,vascular-related risk factor,blood biochemical indicators between PACG group and normal contol group(P＞0.05 ),but a higher intraocular pressure( IOP)was displayed in PACG group compared with normal control group ( P =0.00 ).The numbers of EPCs were ( 48 ± 22 ) cells/ml in PACG group and ( 65 ± 20 )cells/ml in normal control group with a significant difference between them (P=0.004).In PACG group,the numbers of EPCs were(60± 19 )cells/ml and (34 ±7 )cells/ml respeetively in phase 1 and phase 3 of optical nerve damage (Z=-3.015,P=0.002 ).There was a negative correlation between EPCs numbers and baseline IOP within a certain range( r=-0.835,P＜0.05 ).However,no obvious correlations were seen between EPCs numbers and blood lipid Level,blood glucose level or glaucoma course ( r =0.343,P =0.227 ; r =-0.203,P =0.419 ; r =0.198,P =0.610 ).The EPCs numbers in PACG patients with cardiovascular disease were(56±22)cells/ml and that of without PACG were (35± 15 ) cells/ml( P =0.005 ). ConclusionsThe numbers of EPCs decrease in PACG patient.These results imply that EPCs might play role during the restore of the optical nerve damage in PACG eye.

OBJECTIVETo explore the auricular point specificity of the electric determination in stroke so as to provide the evidence for the objective study on the auricular point diagnosis in stroke.

METHODSThirty patients of stroke were selected in an observation group; 30 patients of non-stroke internal medicine disorder were in a control group A; 30 persons of healthy examination were in a control group B. In the observation group.and the control group A, beside the conventional treatment, acupuncture was applied to all the cases. In the observation group, Jianyu (LI 15), Quchi (LI 11), Shousanili (LI 10), Hegu (LI 4), Waiguan (TE 5), Liangqiu (ST 34), Zusanli (ST 36), Sanyinjiao (SP 6) and Taichong (LR 3) were selected on the affected side. In the control group A, Yin-tang (GV 29), Baihui (GV 20), Sishencong (EX-HN 1), Fengfu (GV 16), Dazhui (GV 14), Fengchi (GB 20), Tianzhu (BL 10), Jiaji (EX-B 2) C4-C7 were selected. Acupuncture was given once a day in the two groups. The; treatment of one week made one session, and continuously 2 sessions were required. In the control group B, the convention physical examination was provided without any medication and acupuncture involved. The CLRH-A auricular point detector was used to detect the entire auricular point area in the subjects and the positive reaction sites of auricular points in the subjects of each group before and after treatment separately. The auricular point positive detecting score and positive point frequency were observed in the subjects.

RESULTSBefore treatment, the auricular points detecting score in the observation group was obviously higher than that in the control group A and control group B (both P<0. 05). The difference in the score between the control groups was not significant in' comparison (P>0. 05). In two weeks of treatment, the auricular points detecting score in the observation group was reduced apparently (P<0. 05). The difference was not significant before and after treatment in either the control grolip A or the control group B (both P>0.05). In the observation group, the first 15 points with high frequencies of positive reaction were naogan (AT3,4i), pizhixia (AT4), e (AT1), xin (CO15), gan (CO12), nie (AT2), zhen (AT3), shen (CO10), jiaogan (AH6a), pi (CO13) shenmen (TF4), wei (CO4), neifenmi (CO18), shenshangxian (TG2p) and erjian (HX6,7i). The differences were significant in the frequencies of positive reaction at 9 auricular points as compared with the control group A and the control group B respectively, named naogan (AT3,4i), pizhixia (AT4), e (AT1), xin (CO15), gan (CO12), nie (AT2), zhen (AT3), shen (CO10) and jiaogan (AH6a) (all P <0. 05).

CONCLUSIONThe pathological degree of positive reaction about auricular points in stroke patients is higher than those in the patients of non-stroke internal medicine disease and the healthy people. It is verified that the auricular diagnostic method is specific in stroke. naogan (AT3,4i), pizhixia (AT4), e (AT1), xin (C15), gan (GO12), nie (AT2), zhen (AT3), shen (CO10) and jiaogan (AH6a) are correlative with stroke.

Acupuncture Points ; Acupuncture, Ear ; Adult ; Aged ; Case-Control Studies ; Female ; Humans ; Male ; Middle Aged ; Stroke ; therapy ; Treatment Outcome

Background The study on normal stem cell markers provides a new way of thinking of that pathogenesis of cancer research and looking for specific markers of cancer stem cells.Importin13 (IPO13) is a novel nucleus-cytoplasm transport receptor protein of importin β family,the study on the biological behavior of IPO13 in ocular tissue and limbal neoplasms is lacking.Objective This study was to investigate the differential expression of IPO13 and p63 in human benign and malignant conjunctiva-cornea neoplasms.Methods The specimens of normal donor limbal and conjunctival tissues (6),conjunciva-cornea papilloma (CCP) (6) and conjunctiva-cornea intraepithelial neoplasia(CCIN) (6)were collected from Xiangya Hospital of Center South University.The expressions of IPO13 and p63 in the corresponding tissue were qualitatively and quantitatively detected using immunochemistry.The A values of IPO13 and p63 positive response were calculated and compared among the 3 types specimens.Results The immunohistostaining on frozen sections showed that IPO13 was expressed in nuclei of basal cells of limbal and conjunctival epithelium and the cellular nuclei of basal cells and suprabasal cells of CCP and CCIN epithelium.The A values of IPO13 positive expression were 1687± 1014,3546± 1375 and 7635 ±2854 in the normal limbal specimen,CCP and CCIN,respectively,showing a significant difference among them (F =7.23,P＜0.05),and the A value was higher in the CCP and CCIN than that in the normal limbal tissue (q =4.02,5.13,P＜0.05),and that in the CCIN was significantly elevated in comparison with CCP(q =3.45,P＜0.05).p63 was expressed in nuclei of basal cells and suprabasal of limbal,conjunctival and CCP epithelium,and was expressed in nuclei of the entire CCIN epithelium.The expressions of p63 in the normal conjunctiva-cornea tissue,CCP and CCIN were 2110± 1229,3966± 2129 and 6650± 2136 respectively,with significant difference among the three different specimens (F =6.17,P＜ 0.05),and the A value of p63 positive expression was significantly elevated in the CCP and CCIN compared with normal limbal specimen (q =4.33,5.01,P＜0.05),and that in the CCIN was significantly elevated in comparison with CCP(q=3.83,P＜0.05).Conclusions IPO13 is more specifical in marking the poorly differentiated cells in limbal epithelial proliferative lesions than p63.Compared with the normal limbal specimen,the expressions of IPO13 and p63 in CCP and CCIN specimens gradually upregulat,which suggests that IPO13 and p63 may play a positive regulatory role in conjunctiva-cornea proliferative lesions.The differential expression of IPO13 and p63 is predominant between benign and malignant limbal epithelial proliferative lesions,indicating that IPO13 and p63 may play an important role in regulating the abnormal proliferation and differentiation of limbal epithelial proliferative lesions.

The effects of pretreatment methods of Jerusalem artichoke tubers on microbial lipids fermentation with an oleaginous yeast strain Rhodosporidium toruloides Y4 were investigated in shaking flask culture.The yeast strain accumulated substantial amount of lipids using either purple-or white-skinned Jerusalem artichoke tubers as sole carbon and energy source.When cells were cultured on the extracted juice or the acidhydrolysate,cellular lipid content reached 40%(w/w),while cultured on the pulp,the white-skinned tubershadhigher lipid productivity,yielding 12.1 g lipids per100 g dried tubers.Major fatty acid constituents of microbial lipids were those contained 16-and 18-carbon atoms based on GC analysis,which is quite similar to traditional vegetable oil.Microbial lipids prepared from Jerusalem artichoke can be applied to biodiesel production.

To investigate the effect of Tanreqing injection on immune activity of peripheral blood lymphocytes of patients with lung cancer. The peripheral blood lymphocytes of patients with lung cancer and healthy persons were separated by the density gradient centrifugation method for subsequent experiments, with those from healthy persons as the positive control. The effect of Tanreqing injection on stimulating the proliferation of lymphocytes with phytohemagglutinin (PHA) was determined by MTT method. The effect of Tanreqing injection on the lymphocyte secretions of IFN-γ and TNF-α and the subset ratio of lymphocytes cultured separately or with Tanreqing injection of different concentrations were examined by ELISA and flow cytometry (FCM) respectively. In addition, the LDH release assay was used to detect the cytotoxicity of cytotoxic T cells (CTL) and natural killer cells (NK). According to the findings, all of immunological indexes of lymphocytes from patients with lung cancer were weaker than that of healthy persons, but with the obvious increases in proliferation activity and IFN-γ and TNF-α secretions of lymphocytes co-cultured with Tanreqing Injection (P < 0.05). Among lymphocyte subsets co-cultured with Tanreqing Injection, CD3+, CD3+ CD4+ and CD3- CD16 + 56+ cell ratios notably increased, whereas CD4+ CD25+ Treg cell ratio obviously decreased (P < 0.05). In the meantime, Tanreqing injection can markedly promote the cytotoxicities of CTL and NK (P < 0.05). In conclusion, Tanreqing injection shows a significant effect in promoting the immune activity of lymphocytes from patients with lung cancer and their anti-tumor immunity.
Cell Proliferation ; drug effects ; Cells, Cultured ; Drugs, Chinese Herbal ; administration & dosage ; Humans ; Interferon-gamma ; genetics ; immunology ; Killer Cells, Natural ; drug effects ; immunology ; Lung Neoplasms ; drug therapy ; genetics ; immunology ; physiopathology ; T-Lymphocytes, Cytotoxic ; drug effects ; immunology ; Tumor Necrosis Factor-alpha ; genetics ; immunology

Objective To investigate the prognostic factors of survival time of the Uygur and Han nation-ality elderly patients( over 60 years) with pancreatic cancer in Xinjiang.Methods We carried on a retrospective study of 313 aged patients diagnosed with pancreatic cancer in The First Affiliated Hospital of Xinjiang Medical University from January 1st,2003 to May 30th,2015.We used Kpalan-Meier method for calculation of survival, used Log-rank method for those factors which could affect the prognosis of patients,at last we used Cox propor-tional risk model for those multiple factors which match the role.Results Three hundred and thirteen cases with pancreatic cancer had a median survival of 157 days,and survival rates of half a year,1 and 2 year were 34.8%, 18.5%,7.0%.There was a statistic difference between Uygur patients′and Han patients′survival time(P<0.05).Single factor analysis showed the nationality,tumor size,surgery,ZPS(ECOG),Clinical stage(TNM), chemotherapy and radiotherapy,carcino-embryonic antigen(CEA)level and cancer antigen 199(CA199)level before treatment with outcome(P<0.05).Multiple factor analysis showed that the clinical stage,surgery(P<0.05)can be regarded as independent prognostic decision factors.Conclusion The clinical staging,surgical treatment could be regarded as independent prognostic factors for the elderly prognosis of pancreatic cancer.Both of the Han and Uygur patients,earlier discovered and more appropriate surgery treatment are the key for the elder-ly patients with pancreatic cancer.

ObjectiveTo screen mimetic HIV-1 neutralizing epitopes from plasma with high level neutralizing antibody,and to provide useful information for further study of the interaction between antigen and antibody.MethodsIn order to gain neutralizing antibody recognized mimotopes, we detected neutralizing antibodieslevelsof 11HIV-1infectedslowprogressorsbyPBMC-basedneutralization assays.High-titer HIV-neutralizing antibodies from plasma of SPs was used as the ligand for biopanning by phage-displayed random peptide library.Positive phage clones was evaluated by ELISA,sequenced,and analyzed for homology to HIV-1 env by local BLAST to deduce the neutralizing peptide.ResultsTwenty-two clones were obtained consistent with requirement through three rounds biopanning.After comparison analysis,twelve clones include C8 were obtained as mimotopes of neutralizing antibody,C40 located in gp41Ⅱ cluster with the highest titer by inhibition ratio may be as neutralizing epitope.Conclusion By the use of IgG antibodies from SPs to screen the phage random polypeptide library,one can acquire multiple phage mimetic peptides of HIV related antigen epitope.(Chin J Lab Med,2012,35:838-842 )

10.3969/j.issn.1007-3969.2013.05.008

AIM: To investigate the sensibility of pattern visual evoked potential (P-VEP) on the diagnosis of glaucoma in different temporal frequency.METHODS: The P-VEP were recorded in 51 eyes of 30 primary glaucoma (PG) patients, 16 eyes of 13 ocular hypertension (OHT) patients and 46 eyes of 23 age-equivalent normal people using an array of different temporal frequency and then the patients were compared respectively with the normal group.RESULTS: The P100 wave amplitude in OHT group was significantly lowered at 8Hz, and was lowered at all temporal frequency in PG group, especially when the temporal frequency was higher.CONCLUSION: The P-VEP can be abnormal when the PG is still in its early stage, so it can be an index of early diagnosis and this is more obvious when the temporal frequency is higher, especially at 8Hz.