【Objective】 To investigate the incidence of β-th alassemia (β-thal) heterozygote carrying α-thalassemia (α-thal ) 1 gene in Guangdong area. 【Methods】 β-thal genes were screened by reve rse dot blotting (RDB). In β-thal DNA samples α-thal 1 genes were am plified using gap-PCR method. 【Results】43 α-thal-1 cases were identifi ed among the 500 β-thal traits. The rate is 8.6%. 【Conclusion】 In Guangd ong area the incidence of β-thal heterozygote carrying α-thal-1 gen e is 8.6%, which should be paid much attention in the genetic counselling and pr enatal diagnosis.

The underlying effect of different concentrations of neogenin on proliferation, apoptosis and the related proliferative factors in human trophoblasts was explored in order to understand the function of neogenin during placentation. TEV-1 cell line was cultured and the expression of netrin-1 was detected by using indirect cellular immunofluorescence. Exponentially growing TEV-1 cells were treated by different concentrations of neogenin (0, 1, 5, 10, 50 ng/mL) for 24 h. Cell viability was measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. TEV-1 cell apoptosis was assessed by flow cytometry (FCM). The expression of netrin-1 mRNA and protein in TEV-1 cells was examined by using real-time PCR and Western blot, respectively. It was found that immunoreactivity for netrin-1 was observed in cytoplasm of the trophoblasts. Immediately after treatment with different concentrations of neogenin for 24 h, the netrin-1 expression began to increase. Real-time PCR revealed that the expression level of netrin-1 mRNA was 37.59+/-10.25 times higher than control group when TEV-1 cells were exposed to 50 ng/mL neogenin (P<0.01), and the same tendency was seen by using Western blot. MTT results showed that proliferation of TEV-1 cells was independent of neogenin. Meanwhile, apoptosis was significantly increased to (22.15+/-6.15)% at 50 ng/mL neogenin and (6.55+/-0.25)% without neogenin (P<0.01). It is suggested that neogenin regulates proliferation and apoptosis of TEV-1 cells. And it can enhance the ability of TEV-1 cells to express netrin-1 in a dose-dependent manner. Neogenin may play an important biological role in the normal human pregnancy and contribute to the physiological pregnancy process.

The O152 antigens of Escherichia coli contains a Glc-β-1,3-GlcNAc linkage within the repeating unit. The wfgD gene in E. coli O152 O antigen gene cluster had been demonstrated utilizing NMR technique to encode a glucosyltransferase which is responsible for the synthesis of Glc-β-1,3-GlcNAc linkage. In this study, a synthetic substrate analog of the natural acceptor substrate undecaprenol-pyrophosphate-lipid [GlcNAc-α-PO_3-PO_3-(CH_2)_(11))-O-phenyl]) was used as an acceptor and UDP-Glc as a donor substrate, and electrospray ionization tandem mass spectrometry(ESI-MS-MS) was used for the detailed structural characte-)rization) of the enzyme product. A systematic study was conducted on product to allow rationalization of the fragmentation) processes. The major fragments observed in the ESI-MS-MS spectra result from cleavage of glycosidic) bond and diphosphate moiety. The fragment originating from the nonreducing end of the product yields information on sequence). Cross-ring cleavages, which are very informative of the linkages of the monosaccharide residues constituting) the product, and "internal" cleavage ions which are derived from elimination of substituents from around) the pyranose ring, were also observed. This extensive fragmentation was shown that the expected Glc-β-1,3-GlcNAc linkage in the product, confirming that wfgD is in the form of UDP-Glc: GlcNAc-pyrophosphate-lipid β-1,3-glucosyltransferase.)

Objective To retrospectively study the perioperative complications and postoperative function recovery of 93 patients treated with simultaneous bilateral total hip arthroplasty (THA). Methods A total of 93 patients (186 hips) undergone simultaneous bilateral THA from January 1999 to January 2009 in our hospital were involved in this study. There were 70 males and 23 females (at age range of 25-65 years, average 41. 8 years). The preoperative diagnosis included bilateral avascular necrosis of femoral head in 48 patients, rheumatoid arthritis in 11, developmental dysplasia of the hip in 26 and ankylosing spondylitis in 8. The intraoperative blood loss, Harris scores before operation and at final followup as well as perioperative complications were analyzed. Results All the patients were followed up for average 65 months (12-118 months), which showed femur fracture in one patient and infection six months after discharge in one patient. The Harris score was increased from (36.7 ±6.1) points preoperatively to (91.2±6.2) points at the final follow-up. Hip pain disappeared in 92 patients after operation and radiograph showed no loosening. Actebular loosening occurred in one patient 49 months after operation and was revised accordingly. Conclusion Under strict control of operation indications, suitable choice and implantation of the prosthesis and emphasis on perioperative management and postoperative rehabilitation, simultaneous bilateral THA is a safe and effective choice for bilateral hip diseases.

Objective To build the cohort of drug resistance and analyze treatment efficiency of AIDS patients and situation of drug resistant mutations among HIV-1 infected individuals.Methods A cohort of 116 HIV-1 infected patients was built and their treatment progress were acquired once every 6 months.At the sanle time CD4+ T cell counts and HIV-1 viral load were measured and genotyping for drug resistance was determined by a home brew nested PCR.Results The CD4+ T cell count(470±251/ml)was higher than that before treatment in patients who were treated by AZT/DDI/NVP or D4T/DDL/NVP.The viral load was lower than that before treatmenL The drug resistant mutation frequency increased gradually along with treatment.The CD4+ T cell count was decreased and viral load was increased and the prevalence of drug resistant mutation was increased in the patients who changed regimens to AZT/3TC/NVP or D41/3TC/NVP.Only one primary mutation that was resistant to non-nucleoside reverse transcriptase inhibitors (NNRTIs)was detected in the naive patients.The cross-resistant mutation was detected in two patients after 6 months treatment. The intermediate resistance to lopinavir(LPV) was detected after 12 months treatment.The prevalence of high-grade resistances to NNRTIs was increased obviously,and the prevalence of multi-resistance and cross-resistance was detected in 5 patients after 36 months treatment.Conclusions The prevalence of primary mutation was rare in naive HIV-1 infected patients.The prevalence of drug resistant mutation was inereased gradually along with treatment.Ahhough few regimens were available,the treatment effect could last relatively long period of time if patients keep taking medicine stably.The regimens could be changed according to the results of drug resistant test.

OBJECTIVETo study the inhibition of berberine (BBR) against ECV-304 apoptosis induced by Staphylococcus aureus (S. aureus).

METHODSECV-304 cells were pre-treated with 128 microg/mL BBR for 2 h and then S. aureus was added (1:100). The viability of cells was detected by MTT (3-4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The morphological changes were observed by Hoechst 33258 staining. The protection of BBR for infected cells was detected by DNA Ladder.

RESULTSECV-304 cells' viability were not obviously affected by berberine. But S. aureus induced ECV-304 cells' viability could be significantly inhibited by pre-treatment of BBR (P < 0.05). Besides S. aureus-induced ECV-304 apoptosis could be reduced, with significantly lessened apoptotic body and unobvious DNA degradation.

CONCLUSIONBBR could significantly inhibit S. aureus induced ECV-304 apoptosis.

Apoptosis ; drug effects ; Berberine ; pharmacology ; Cell Line ; Human Umbilical Vein Endothelial Cells ; drug effects ; microbiology ; pathology ; Humans ; Staphylococcus aureus

Background Leber hereditary optic neuropathy (LHON) is mitochondrial DNA (mtDNA) disease and mainly leads to optical nerve degeneration.Its primary mechanism is synthesis disorder of DN4 protein due to variation of mtDNA 11778 locus.So to construct a vector with exogenous normal ND4 and transfect into mitochondria is a key of gene therapy for LHON.Objective This study was to investigate the in vitro transfection of adeno-associated virus (AAV)-ND4 gene into mitochondria.Methods Human renal epithelial cell lines transfected adenovirus E1A (293 cells) were regularly cultured and divided into two groups.Framework plasmids of recombinant AAV-ND4 or simple AAV2 were added to the cell medium respectively.The expression of ND4 in cells were located 12,24,36 and 48 hours after transfected by Y03 dual fluorescent quantum dots staining.The positive response for ND4 showed the green fluorescence.Results Cultured 293 cells grew well with 80％ confluence.Abundant green fluorescence particles were seen in cytoplasm in the AAV-ND4 transfected group,but only red fluorescence from mitochondrial protein was seen in the simple AAV transfected group under the fluorescence microscope.Conclusions Exogenous ND4 protein can been successfully transfected into mitochondria using the ND4 gene constructed AAV.This result provides experimental evidence for the further study on gene therapy of LHON.

Objective To observe the effects of the drug pair of Rhizoma Polygoni Cuspidati ( Huzhang) and Ramulus Cinnamomi ( Guizhi) on the Toll-like receptor 4 mediated myeloid differentiation factor 88 ( TLRs/MyD88) signaling pathway of rats with acute gouty arthritis induced by monosodium sodium urate (MSU) , so as to explore its therapeutic mechanism. Methods Forty-eight male SD rats were divided into normal group, modele group, blank plasmid group, positive plasmid group, Huzhang- Guizhi herb-pair (7 g/kg) group, and Huzhang-Guizhi herb-pair ( 7 g/kg) siRNA group, 8 rats in each group. The normal group, plasmid groups and model group were given physiological saline, and the left groups were given the corresponding drug by intragastric administration for 10 continuous days ( once daily ) . On the seventh day of intragastric gavage, acute gouty arthritis were induced by injection of MSU into the rat ankle joint, and normal group was injected with the samevolume of normal saline. Positive plasmid group and Huzhang-Guizhi herb-pair siRNA group were injected with the constructed siRNA-TLR4 plasmid targeting TLR4 gene ( TLR4-siRNA) to inhibit the in-vivo TLR4 gene expression. Pathological changes of the synovial tissues were detected, the contents of peripheral blood tumor necrosis factor alpha ( TNF-α) and interleukin 1 beta ( IL-1β) were detected by double antibody sandwich method, and the mRNA and protein expression levels of TLR4, MyD88, TNF receptor-associated factor 6 ( TRAF-6) in peripheral blood mononuclear cells of rats were detected by real-time fluorescence quantitative polymerase chain reaction ( PCR) and Western blot methods. The nuclear factor kappa B ( NF-κB) p65 immunoactivity was assayed by immunohistochemistry. Results Compared with the normal group, the model group had obvious hyperplasia of synovial cells and the inflammatory cell infiltration ( dominated by lymphcytes and monocytes) , and had amount of cellulose adhesive on the synovial membrane surface. Compared to the model group, positive plasmid group, Huzhang- Guizhi herb-pair group and Huzhang-Guizhi herb-pair siRNA group could obviously relieve the inflammatory cell infiltration, and improve synovial cell proliferation reaction. Compared to the normal group, serum levels of TNF-α and IL-1β, and the expression levels of TLR4, MyD88, TRAF-6 mRNA and protein in the peripheral blood mononuclear cells as well as the synovial NF-κB p65 ex pression in the model group were significantly increased ( P<0.01). Compared to the model group, positive plasmid group, Huzhang-Guizhi herb-pair group and Huzhang- Guizhi herb-pair siRNA group showed significant decrease in the levels of TNF-α, IL-1β, TLR4 MyD88, TRAF-6 and NF-κB p65 ( P<0.05 or P<0.01) . Conclusion Huzhang-Guizhi herb-pair can regulate the cytokines of the synovial membrane tissue in acute gouty arthritis rats, which may be related with its effect on inhibiting abnormal activation of TLR4-MyD88-NF-κB pathway in synovial tissue.

OBJECTIVETo investigate the effect of curcumin on the injury in hippocampal neurons and the expression of regulated upon activation nonnal T-cell expressed and secreted (RANTES) in hippocamp during cerebral ischemia/reperfusion (I/R) in rats with spontaneous hypertension (SH).

METHODSMale Wistar-Kyoto (WKY) rats and spontaneous hypertension rats (SHR) were randomly divided into five groups (n = 6): sham group (W-Sham and S-Sham group), ischemia/reperfusion group (W-/R and S/R group), curcumin group (S-Cur group) . Each group was splitted into 5 subgroups of 3 h,12 h, 1 d, 3 d and 7 d according to the time interval before reperfusion. Global brain ischemia/reperfusion model was established by 4-VO method. Hematoxylin-eosin staining (HE staining) was used to observe the vertebral cell morphology in hippocampal CA1 region. Nissl staining was applied to detect the average density of cone cells in hippocampal CA1 region. The expression of RANTES in hippocamp was determined by ELISA. The behavior of the rats was evaluated at 7 days after reperfusion. Results: Compared with the sham group rats, the ability of learning and memory was significantly decreased in ischemia/reperfusion group rats, the number of injured neurons were greatly elevated , the protein expression levels of RANTES was significantly increased (P < 0.05). Compared with W-I/R group rats, the ability of learning and memory in S-I/R group rats was greatly reduced, the number of injured neurons increased extremely, the protein expression level of RANTES was significantly enhanced( P <0.05). The number of injured neurons declined significantly in S-Cur group rats, the ability to learn and remember of these rats was improved and the RANTES protein content decreased significantly (P < 0.05).

CONCLUSIONSHR are more susceptible to ischemia/reperfusion induced hippocampal neuronal injury which may be improved by curcu min. Its underlying mechanism is possibly associated with the inhibition of RANTES protein expression level.

Animals ; Brain Ischemia ; metabolism ; pathology ; physiopathology ; Chemokine CCL5 ; metabolism ; Cognition ; drug effects ; Curcumin ; pharmacology ; Hippocampus ; cytology ; metabolism ; pathology ; Hypertension ; metabolism ; pathology ; physiopathology ; Male ; Neurons ; drug effects ; metabolism ; pathology ; Rats ; Rats, Inbred SHR ; Rats, Inbred WKY ; Reperfusion Injury ; metabolism

BACKGROUND: Immunoloregulation of mesenchymal stem cells (MSCs) is commonly approved. Previous studies have confirmed the ability of Flk-1~+ bone marrow MSCs (BMSCs) to inhibit T/B lymphocyte proliferation in vitro. OBJECTIVE: To investigate the therapeutic effect of Flk-1~+ BMSCs in collagen-induced arthritis mice.METHODS: A total of 18 healthy male DBA-1(H-2K~q) mice aged 10 weeks were randomly divided into 3 groups. All the mice were injected at the base of the tail with bovine type II collagen (CII), and received a booster injection of CII on day 21 to establish the CIA mice model. DBA-1(H-2K~q)mouse Flk-1~+ BMSCs were isolated in vitro by the density gradient centrifugation and adherence screening. Following initial immunity, mice in the cell transplantation group were infused with Flk-1~+ BMSCs (1-2)×106 cells/mouse via the caudal vein. Mice in the cell transplantation group were injected with the same volume of Flk-1~+ BMSCs during booster. Mice in the model control group were injected with an equal volume of saline 0 or 21 days following initial immunity. Following initial immunity and booster immunization, claw pad thickening and clinical score were observed, changes of joint pathology and dynamic changes in serum factor mass concentration were determined in mice. RESULTS AND CONCLUSION: Compared with the model control group, no significant difference in claw pad thickening and mean clinical score was detected in the cell transplantation group following initial immunity (P > 0.05), with the presence of obvious damage to synovial membrane and inflammatory cell infiltration. Mass concentration of each serum cell factor was similar. The claw pad was significantly thickened (P < 0.01), mean clinical score reached 3.35 points, with severe damage to synovial membrane, proliferation of blood capillary in the cell transplantation group following booster immunization. Interleukin-6 levels were greatly increased at day 28 following initial immunity (P < 0.1), but decreased at day 35 following initial immunity (P < 0.1). Results indicated that in the collagen-induced arthritis mouse models, Flk-1~+ BMSC transplantation did not obtain prospective therapeutic efficacy, but aggravation of arthritis was observed in the cell transplantation group following booster immunization. Upregulation of interleukin-6 concentration could aggravate the behavior symptom of rheumatoid arthritis mice.