Objective To investigate the protective effect of indole-3-carbinol (I3C) on radiation-induced mouse bone marrow hematopoietic cell injury and the involved mechanisms. Methods (1) The bone marrow nuclear cells (BMNCs) from CD45.1 subtype of C57BL/6J mice were collected by a density gradient centrifugation method. The BMNCs were pretreated with a series doses of I3C (0 mol/L, 10-8 mol/L-10-3 mol/L) and then exposed with radiation of 137Csγ-ray (doses of irradiation were 0 Gy, 1 Gy and 4 Gy). After 18-hour culturing, the bioluminescence method was used to detect the cell viability. (2) These cells were divided into control group and 10-6 mol/L I3C group. Both groups were received the irradiation (0 Gy, 1 Gy and 4 Gy) and inoculated into the methylcellulose semi-solid culture medium to incubate 7 days, the colony forming unit-granulocyte monocytes (CFU-GM) were observed. (3) Twenty-four CD45.2 subtype mice used as the receptor were exposed with 8 Gy radiation. The CD45.1 BMNCs were divided into control group, 4 Gy irradiation group, 4 Gy irradiation and 10-6 mol/L I3C group. Donor cells were harvested from C57BL/6J (CD45.1) mice after they received various treatments, and were then mixed with competitive BMNCs from C57BL/6J (CD45.2) mice. The mixed cells were transplanted into recipient mice (8 mice/group). Flow cytometry was used to analyze the proportion of donor cells in peripheral blood of receptor. (4) The cells were divided into control group, 10-6 mol/L I3C group, 1 Gy irradiation group, 1 Gy irradiation with 10-6 mol/L I3C group. After 24-hour culturing, Western blot assay was used to detect the expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and hemeoxygenase-1 (HO-1). Results (1) I3C showed a significant cytotoxic effect on the BMNCs when its concentration was above 10-4 mol/L. 10-7-10-6 mol/L I3C could reduce the radiation injury of BMNCs under the same dose of irradiation. Therefore, 10-6 mol/L I3C was chosen for subsequent experiments. (2) The CFU-GM was significantly higher in 10-6 mol/L I3C group than that of control group (P<0.05). (3) Results of flow cytometry showed that the proportion of donor cells in receptor was significantly higher in 4 Gy irradiation group than that of control group, which decreased the engraftment capability of irradiated HSCs (P<0.05), although the engraftment capability of irradiated HSCs improved after 10-6 mol/L I3C treatment. (4) I3C significantly enhanced the increased protein expression of Nrf2 and HO-1 caused by radiation (P<0.05). Conclusion I3C has a protective effect on hematopoietic cells following radiation-induced injury, which may be related with activating the Nrf2/HO-1 signal pathway.

Objective To investigate the protective effect of 3,3′-diindolylmethane (DIM) on radiation-induced injury in mouse hematopoietic system. Methods Thirty C57BL/6 mice were randomly divided into control group, 2 Gy irradiation group and 2 Gy irradiation+DIM group (n=10 for each group). Mice of control group received sham irradiation, and the other two groups accepted 2 Gy 137Cs γ-ray total body irradiation. Mice in 2 Gy irradiation +DIM group were intraperitoneally injected 75 mg/kg DIM 30 min before irradiation. Mice of other two groups were treated with reference solution. After 7 d and 15 d of 2 Gy irradiation, the peripheral blood samples were collected to count the number of bone marrow nuclear cells (BMNCs). The level of reactive oxygen species(ROS) was measured by DCFH-DA. The levels of colony forming units-granulocyte-macrophage (CFU-GM) were also detected. Results The numbers of white blood cell (WBC), platelet count (PLT), BMNCs and CFU-GM were significantly decreased and the ROS level of bone marrow cells increased significantly in the irradiated group than those of control group (P<0.05). Compared to 2 Gy irradiation group, the numbers of WBC, PLT, BMNCs and CFU-GM were significantly increased in 2 Gy irradiation +DIM group, and the level of ROS was decreased significantly (P<0.05). Conclusion DIM has a protective effect on hematopoietic cells following radiation-induced injury, which may be related with the decreased ROS level.

OBJECTIVETo compare the clinical efficacy between acupuncture and intravenous administration of lipoic acid and alprostadil for distal symmetric multiple peripheral neuropathy of diabetes mellitus.

METHODSSixty patients were randomly divided into an acupuncture group (31 cases) and a medication group (29 cases). Patients in the two groups received basic treatment to control blood sugar within a safe range. On this basis, patients in the acupuncture group were treated with acupuncture at Geshu (BL 17), Weiwanxiashu (EX-B 3), Ganshu (BL 18), Pishu (BL 20), Shenshu (BL 23), Zusanli (ST 36), Taixi (KI 3), ashi points, etc. Patients in the medication group were treated with intravenous administration of lipoic acid (0.6 g) and alprostadil (10 mg). The treatment was given once a day, 10 days for a course of treatment; there was an interval of 2 days between courses, and totally 3 courses were given. The score of peripheral neuropathy of diabetes mellitus, the change of nerve conduction: velocity and clinical efficacy before and after treatment in the two groups were observed.

RESULTSAfter treatment, the score of peripheral neuropathy was significantly reduced in the two groups (both P < 0.05), which was more significant in the acupuncture group (P < 0.05). After treatment, the sensory nerve conduction velocity (SNCV) and motor nerve conduction velocity (MNCV) of median nerve and common peroneal nerve were significantly increased in the two groups (all P < 0.05). Compared between two groups, the SNCV and MNCV of common peroneal nerve in the acupuncture group were significantly superior to those in the medication group (both P < 0.05); the MNCV of median nerve in the acupuncture group was significantly superior to that in the medication group (P < 0.05); the SNCV of median nerve in the acupuncture group was not significantly different from that in the medication group (P > 0.05). The total effective rate was 83.9% (26/31) in the acupuncture group, which was significantly superior to 62.1% (18/29) in the medication group (P < 0.05).

CONCLUSIONAcupuncture and conventional medication both have satisfied effects for distal symmetric multiple peripheral neuropathy of diabetes mellitus, and acupuncture is superior to medication on improving clinical signs of sensory disorder, reflection disturbance and muscle weakness, nerve conduction and clinical curative effect.

Acupuncture Points ; Acupuncture Therapy ; Adult ; Aged ; Diabetes Mellitus, Type 2 ; complications ; Diabetic Neuropathies ; etiology ; physiopathology ; therapy ; Female ; Humans ; Male ; Middle Aged ; Peripheral Nerves ; physiopathology ; Peripheral Nervous System Diseases ; etiology ; physiopathology ; therapy

Objective To analyze the resenilin-1 (PS-1) gene mutations in Alzheimer' s disease (AD) patients and investigate the influence of the initiation codon mutation on the mRNA expression of PS-1 and amyloid precursor protein (APP) genes and the expression of PS-1 proteins.Methods (1) All 111 AD patients were enrolled by the Department of Neurology,Second Affiliated Hospital,College of Medicine,Zhejiang University from July 2004 to June 2010.Mutations in the 13 exons and flanking regions of PS-1 gene were examined by direct sequencing.(2) cDNAs encoding full-length wild-type and mutant (c.1A ＞G) PS-1 were subcloned into enhanced green fluorescent protein.Levels of the mRNA expression of PS-1 and APP genes and PS-1 proteins expression in the transfected cells were detected by quantitative real-time PCR and Western blot,respectively.Results A new heterozygous initiation codon mutation changing from ATG to GTG in one individual was identified.Compared to the control groups,the mRNA expression of the mutant PS-1 gene in HEK293 and N2a was significantly lower than the normal PS-1 gene(116.8 ± 3.9 vs 49.5 ±3.3,t =13.27,P ＜0.01 ;69.0 ± 1.9 vs 29.5 ± 1.3,t =17.20,P ＜0.01) and the APP gene was not obviously altered.The proteins were detected by Western blot analysis in HEK293 cells but not in N2a cells.Conclusions Since we only identified one novel heterozygous initiation codon mutation (from ATG to GTG),mutations in PS-1 are likely to be rare in AD patients.Initiation codon mutation would reduce the expression of PS-1 proteins.Inactivation of some of the PS-1 proteins could be insufficient to lead to AD and could be more likelv to act as a risk factor.

Objective To study the safety and efficacy of low dose Peginterferon-alpha-2a (PEG-INF-α-2a) combined with Ribavirin treating chronic hepatitis C in renal transplant recipients.Methods A total of 13 cases of HCV hepatitis were randomly divided into treatment and control groups.Seven cases in treatment group were given PEG-INF-α-2a (90 μg/week) and ribavirin (600mg/day) for 16 to 48 weeks,and the rest 6 cases in control group were subjected to general liver protection and anti-inflammatory treatment. All patients were followed up for more than 2 years.Results There were 5 cases getting early response in treatment group for 16 weeks,including four cases of complete response and no non-effects response patients. In 4 cases voluntarily receiving treatment for 48 weeks,1 case had facial muscle myalgia and increased Cr level at 35th week,humoral graft rejection was confirmed pathologically,and the treatment was terminated; 1 case had recurrence of HCV RNA replication and PEG-INF-α-2a was withdrawn at 38th week.As results,5 patients in the treatment group obtained complete response after two years,including 2 cases whose HCV-IgG had got negative,HCV RNA replication was significantly lower than in the control group,and the average Cr higher than in control group (P＞ 0.05). There were adverse reactions during this treatment protocol: fever,muscle myalgia,agranulocytosis, anemia and humoral graft rejection.Conclusion The efficacy of low lose PEG-INF α-2a combined with ribavirin is definite in the treatment of chronic HCV hepatitis in kidney transplant recipients.The 16-week treatment duration is reasonable.It is remarkable that PEG-INF-α-2a may cause humoral graft rejection and Cr crawling.

Objective To investigate the compensatory changes in morphology,function,and hemodynamic indices of the retained kidney after nephrectomy among living related donors.Method The 170 living related kidney donors underwent assessments before surgery as well as at 1st and 12th month,postoperatively,including length,width,short diameter,glomerular filtration rate (GFR),effective renal plasma flow (ERPF),peak systolic blood flow velocity (Vsmax),resistance index (RI),as well as pulsatility indices (PI) of main renal artery (MRA),segmental renal artery (SRA),and interlobar renal artery (IRA).Results All subjects were followed up for 9 to 68 months,with no observed hypertension or kidney failure.The length,width,and short diameters of the retained kidney were increased significantly (P＜0.01) at 1st and 12th month postoperatively.The renal sizes at 1st month postoperation were similar to that at 12th month postoperation (P＞0.05).GFR and ERPF were increased significantly as compared with preoperative values (P＜0.01) with similar values at postoperative month 1 and 12 (P＞0.05).The Vsmax of MRA,SRA,and IRA in the retained kidney were increased significantly (P＜0.01),and the RI and PI were also increased as compared with the preoperative values (P＜0.05),albeit these indicators were similar at postoperative months 1 and 12 (P＞0.05).Conclusion For all subjects studied after unilateral nephrectomy in a living related donor,the diameter of the retained kidney as well as the GFR and ERPF showed compensatory increases.Various arterial hemodynamic parameters also showed compensatory changes.Under strict donor inclusion criteria,living related kidney donor procedures is safe.

Objective To express and purify the pneumococcal surface adhesin A(PsaA) protein,discuss its application as a protein carrier in conjugates vaccine. Methods The gene encoding for the PsaA protein was amplified from the genomic DNA of Streptococcus pneumoniae using PCR. The PCR product was then cloned into the prokaryotic expression vector pET-28a and the recombinant was transformed into host cell E. coli BL21 (DE3). The expression of the recombinant protein(rPsaA) was induced by IPTG and purifled by using DEAE anion-exchange chromatography. The rPsaA was successfully conjugated with group A meningococcal polysaccharide(GAMP). The mice were immunized subcutaneously with the conjugate and the immune responses against GAMP and PsaA were detected by ELISA. Results The recombinant PsaA was expressed as a 37 × 103 soluble protein without His-Tag. The rPsaA was successfully conjugated with GAMP. In addition to the immune response against PsaA, The antibody response against GAMP was significant improved in the mice immunized with conjugate vaccine in comparison with those immunized with GAMP alone. Conclusion The recombinant protein PsaA without His-Tag was obtained and conjugated with GAMP. The strong antibody responses against PsaA and CAMP were obtained in the immunized mice at the same time which may provide the protection against pneumonia and meningitis simultaneously.

Objective To investigate the differences in susceptibility to Lewis lung carcinoma and T lymphocyte subsets in the immune microenvironment between young and elderly mice.Methods Six C57/B6 mice at two months(young)and six mice at twelve months(aged)were injected with Lewis lung carcinoma cells at the dose of 1 × 106 in the left armpit to establish a murine model of lung carcinoma.The weight and tumor growth were monitored.Blood samples for routine blood tests were collected after 24 days.The proportions of CD4+ and CD8+T cells in the spleen were detected by flow cytometry,and the infiltration of CD4+,CD8+ T cells and related effector T cells in the tumor microenvironment were determined in the same way.Results The body weight of tumor bearing mice in the aged group was significantly higher than that in the young group(P ＜0.001);The tumor weight in the aged group(5.084±0.528)g was significantly higher than that in the young group(2.963 ±0.378)g(t =3,349,P =0.012);Routine blood tests showed that the numbers of leukocytes and subsets(except mononuclear)in the aged group were significantly lower than in the young group(P ＜0.05);Flow cytometry found that the effector and memory/effector CD4+T cell ratios in the spleen were significantly higher in the aged group than in the young group(P ＜0.001)and the expression of effector and memory/effector CD8+T cells in the tumor microenvironment was also significantly higher than in the young group(P ＜0.05);Quantitative expression values of IL-6 and IL-10 in the tumor microenvironment were 25090±3820 and 10670± 1793 in the aged group and 6252±864 and 3061±451 in the young group,respectively.Moreover,the expression levels of IL-6 and IL-10(t =3.925,P =0.01;t =3.552,P =0.02)in the tumor microenvironment in the aged group were significantly lower than those in the young group.Conclusions Young mice are more susceptible to Lewis lung carcinoma,probably as a result of differences in inflammation and immunity.

BACKGROUND:In recent years, some studies have demonstrated that ganglioside can promote survival and differentiation of umbilical blood cord mesenchymal stem cel s in vitro. OBJECTIVE:To observe the effect of injection of human umbilical blood cord mesenchymal stem cel s and ganglioside into rat lateral ventricles on neurological functional recovery from cerebral palsy. METHODS:Total y 60 cerebral palsy neonatal rats were delivered from pregnant rats which were modes were given intraperitoneal injection of lipopolysaccharide for 2 successive days on day 17 of gestation. Then those neonatal rats were randomly divided into five groups, including model group (n=10), sham transplantation group (n=10), stem cel transplantation group (n=18), ganglioside group (n=10) and combination group (n=12). Under stereotaxic instrument, umbilical blood cord mesenchymal stem cel s or ganglioside were injected into left lateral ventricles of the rat brain, respectively, and the sham transplantation group was given the same volume of phosphate buffered saline. Two rats from the stem cel transplantation group were put to death for immunofluorescence staining at 7, 14, 21 and 28 days after transplantation, respectively, and two rats in the combination group were kil ed for immunofluorescence staining at 14 days. Besides, al rats were underwent neurologic evaluation at 28 days after transplantation. RESULTS AND CONCLUSION:The umbilical blood cord mesenchymal stem cel s could survive, migrate and differentiate, which mainly distributed in the lateral ventricle, hippocampus and cortex. At 14 days after transplantation, positive expressions of BrdU and glial fibril ary acidic protein in the combination group were significantly higher than those in the stem cel transplantation group (P<0.05). In addition, compared with the model group, the holding time significantly prolonged and foot error times significantly decreased in the latter three groups (P<0.05), as wel as in the combination group compared with the stem cel transplantation and ganglioside groups (P<0.05). These results indicate that umbilical blood cord mesenchymal stem cel s and ganglioside can both improve neurological function of rats with cerebral palsy. Given that ganglioside can promote survival and differentiation of umbilical blood cord mesenchymal stem cel s in vivo, the combined transplantation is preferred.

Objective To explore the correlation between circadian clock gene clock circadian regulator (CLOCK) and attention-deficit/hyperactivity disorder (ADHD) and the role of CLOCK and sleep problems on inhibition in male children with ADHD. Methods Two single nucleotide polymorphisms (SNPs) of CLOCK were genotyped in 854 male ADHD children and 320 male controls. Sleep problems were assessed using parent symptom questionnaire. In ADHD cases, the main effects and interaction of CLOCK SNPs and sleep problems on inhibition assessed by using Stroop Color and Word Test, were analyzed using the analysis of covariance (ANCOVA). Results No significant differences of allele and genotype frequencies were found for rs6832769 and rs11932595 in all case-control groups (P>0.05). In ADHD cas?es, the main effects of rs6832769 and rs11932595 genotypes and sleep problems on inhibition were not significant (P>0.05). However, the interaction of rs6832769 genotype and sleep problems was significant (F=6.71, P=0.01). When ac?companied with sleep problems, ADHD cases carrying the AA&AG genotype showed the longest time of word interfer? ence (F=6.63, P=0.01). Conclusions Inhibition of male ADHD children can be modulated by the interaction of CLOCK rs6832769 and sleep problems.