BACKGROUND:Clinical application of neural stem cells is under exploration.Currently,the indicative differentiation of neural stem cells into specific neurons to replace lost and degenerative neurons needs to solve.OBJECTIVE:To explore the effect of 83 ku protein in rat hippocempi on inducing neural stem cell differentiation into acetylcholine esterase (ACHE) positive neurons.DESIGN,TIME AND SETTING:In vitro controlled observation of cytology was performed at the Medical College of Nantong University between October 2003 and April 2008.MATERIALS:A total of 12 SD rats,of clean grade,and SD fetal rats,aged 17 days,were provided by the Experimental Animal Center of Nantong University.METHODS:The normal hippocampi and hippocampi on the 14th day after the hippocampal fimbria transection were prepared into homogenate used for 10% native-polyacrylamide gel electrophoresis,and the differential proteins of 83 ku were electroeluted.The protein concentration was adjusted to 300 mg/L.The forebrain tissues of fetal rats were harvested and neural stem cells were isolated and in vitro cultured:blank control cells were cultured in serum-free DMEM/F12 medium;83 ku normal and 83 ku transection groups were separately cultured in serum-free DMEM/F12 medium containing 10 mg/L 83 ku protein from normal and hippocampal fimbria transection rats for 12 days.MAIN OUTCOME MEASURES:AChE histochemical staining was used to detect the differentiation of neural stem cells into AChE positive neurons.RESULTS:After 12 days of culture,there was a large amount of AChE positive neurons in 83ku transection group and their bodies were very big and the processes were abundant;The AChE positive neurons in 83ku normal group were less than 83 transection group,and their bodies were small with short processes.A few of AChE positive neurons were seen in control group.There were significant differences in number of AChE positive neurons among three groups (P＜0.05).CONCLUSIONHippocampal 83 ku protein can induce neural stem cells to differentiate into AChE positive neurons.

Objective To observe functional changes of renal tubular epithelial cells stimulated by high mobility group protein box 1 (HMGB1) and associated mechanism.Methods Renal tubular epithelial cells (NRK52E) were divided into control group,HMGB1 group and HMGB1+ lipopolysaccharide from Rhodobactersphaeroides (LPS RS) group.Toll-like receptor 4 (TLR4) expression was detected by immunofluorescence and Western blotting.Apoptosis rate and cell cycle arrest were identified with flow cytometry.The activation of MAPK signaling pathway and NF-κB were detected by Western blotting.The IL-1,IL-6 and tissue inhibitor of metalloproteinases 2 (TIMP2) mRNA levels were measured by real-time PCR.The secretion levels of IL-1,IL-6 and TIMP2 were measured by protein chips assay.Results TLR4 was expressed by NRK52E cells.Compared with the control group,there were increased cell cycle G1 arrest,MAPK signaling pathway and NF-κB activation in HMGB1 group.Furthermore,IL-1,IL-6 and TIMP2 mRNA levels were increased and IL-1,IL-6 and TIMP2 were secreted by NRK52E when stimulated with HMGB1 (all P ＜0.05).However,effects mediated by HMGB1 stimulation could be inhibited by LPS RS (all P＜0.05).Conclusions Inflammatory activation of NRK52E cells can be mediated by the interaction of HMGB1 and TLR4.

Objective: To analyze the prognostic factors of non-Hodgkin's lymphoma(NHL)and to investigate the prognostic value of peripheral blood absolute lymphocyte count(ALC)at admission for patients with NHL. Methods: Clinical features and follow-up data of 108 patients with pathologically confirmed NHL seen in our hospital between January 2000 and January 2008 were reviewed.SPSS14.0 package was used for statistical analysis.Kaplan-Meier was applied to assess the survival probability.All parameters statistically significant concluded by univariate analysis were then computed as co-variates for multivariate analysis with Cox regression model. Results: The ratio of males to females was approximately 1.5:1.The median age of patients was 48 years.Before treatment.the Ann Arbor clinical classification showed that 61.1% of the cases were of stage Ⅰ and Ⅱ.Approximately 93%of the patients had ECOG performance status(PS)score of 0-1 and 19.2%of the cases had elevated serum lactate dehydrogenase(LDH).According to intemational prognosis index score.80.6%of the patients were in a low risk group.At admission，35.2%of the cases had ALC≤1×10~9/L.Hemoglobin (Hb)≤110g/L and B symptoms were seen in 29.6%and 26.9%of the patients.The mean Hb was 129.2±17.5g/L in cases with ALC>1×10~9/L(n=70)and 98.1±20.6g/L in cases with ALC≤1×10~9/L(n=38),with a statistically significant difference between the two groups(P<0.05).With a median follow-up duration of 2 years，the median overall survival(OS)time was 2.3 years for all patients.The 2-year and 5-year OS rates were 73.2%and 39.6%，respectively.ALC≤1×10~9/L,Hb≤110g/L,B symptoms and intemational prognostic index(IPI)≥2 were statistically significant unfavorable prognostic factors for NHL revealed by univariate analysis.Multivariate analysis showed that ALC≤1×10~9/L,B symptoms and IPI ≥2 were statistically significant unfavorable prognostic factors for NHL. Conclusion: ALC and B symptoms may be prognostic factors independent of IPI for NHL.Evaluation of the prognosis with IPI,ALC，and B symptoms is of clinical value for individualized therapy of NHL patients.

Objective To investigate the effect of extracellular signal-regulated kinases(ERK) signal transduction in the process of NSCs differentiating into neurons in the fimbria-transected hippocampi's extracts. Methods Twelve Sprague-Dawley rats'right fimbrias were transected. The extracts were gained from the fimbria-transected hippocampi at the 14th day normal rat, and the extracts supernatant fluid was collected after centrifugal process, then the protein concentration in the extracts was determined. In the serum-free medium,NSCs from the fetal hippocampus were planted on 24 well culture plate, then were divided into three group and eight wells for each group as follows: the transected group contained the extracts of the fimbria-transected hippocampi;the normal group contained the extracts of the normal hippocampi;the pure control group have no extracts. After cultured for 14 days,the cells were detected by using MAP-2 and p-ERK immunofluorescence. Result The number, area, perimeter of MAP-2 positive neurons were all declined in transected group, the normal group and the control group orderly. Statistic results showed significant difference between every two groups. The number of MAP-2/p-ERK double-positive neurons were decreased in transected group, the normal group and the control group orderly, but the percentage of double-labeled neurons in total MAP-2 positive neurons were increased in turn. In these two aspect, there were also significant difference between every two group. And most of the MAP-2/p-ERK double-positive neurons were immature. Conclusion The extracts of the fimbria-transected hippocampi had obvious effects on promoting NSCs differentiating into neurons and speeding up the maturation of neurons than those of the normal hippocampi. The morphological results showed that ERK signal transduction might be related to the differentiation of NSCs into neurons.

Objective Modified the medium that can increase single\|clone formed rate and confirmed the single clone spheres had the multipotential of differentation. Methods We modified the medium, that is, the medium contained half of primary culture medium and half of fresh culture medium. A great deal of neurospheres dervied from a single cell were plated averagely into 24 well plates and added into the DMEM differentiation medium (containing serum). After culturing for 14 days, cultures were stained with the neuronal\| ang glial\|specific markers (MAP\|2 for neurons, GFAP for astrocytes and CNP for oligodendrocytes). Results Each 96 well plate containing half of primary culture medium generated two to three single clone spheres, in control plate containing only fresh medium generated half to one single clone sphere. After differentiation, these cell clones expressed MAP\|2, GFAP and CNP positive respectively.Conclusion\ Using half of primary culture medium can increase single\|clone formed rate and these cell clones had the multipotential of differentiation.\;[

Objective To evaluate the effects of ginkgolide B on inducing neural stem cells differentiating into neurons. Methods A great deal of single cell clone neurospheres raised from single cell and proliferated by the technology of serum\|free culture and single cell clone.Suspensions of cell clone neurospheres were plated equably into 24 well plates and added into the 10% FBS differentiation medium containing ginkgolide B,BDNF or without any factor.Cultures were terminated at 7 and 14 days respectively.MAP\|2,neuron\|specific marker,were used to mark neurons by immunofluorescence.MAP\|2 positive neurons were observed and counted by fluorescence microscope.The area and perimeter of these positive neurons were analyzed. Results The number of MAP\|2 positive neurons in ginkgolide B group is more than that in the control group in two periods.The area and perimeter of MAP\|2 positive neurons in ginkgolide B group were markedly larger than those in the control group at 7 and 14 days after cultured,but it's less than those in the BDNF group.Conclusion\ Neural stem cells can be induced to differentiate into neurons by ginkgolide B which has the similar role to BDNF.\;[

Objective To investigate the relationship between the nerve growth factor ( NGF ) induced hippocampal neuroregeneration and homeobox gene Lhx 8.Methods Seventy-two SD rats were divided into control group , transected group, NGF group, transected combined with NGF group after right fimbria-fornix transection and NGF intracerebroventricular injection . Real-time PCR and Western blotting were applied to detect the gene and protein expression of Lhx8 in each group.The choline acetyltransferase ( ChAT)/Lhx8 double labeled cells in subgranular zone ( SGZ) of hippocampus in each group were detected by immunofluorescence .Results The expression of Lhx8 gene and protein in the transected , NGF group and especially in the transected combined with NGF group was obviously higher than in the control group .The number of ChAT/Lhx8 double labeled cells in the NGF group and the transected combined with NGF group was obviously more than in the control group and transected group . Conclusion The hippocampal neuroregeneration which induced by NGF intracerebroventricular injection was associated with the higher expression of Lhx8.

Objective To observe the effect of JLP deficiency on the progression of renal interstitial fibrosis in mice model of unilateral ureteral obstruction (UUO),and to investigate the role and underlying mechanism of JLP in the development of renal fibrosis in obstructive nephropathy.Methods jlp Wild type (jlp+/+) and jlp deficient (jlp-/-) mice were divided into four groups:jlp+/+-and jlp-/--sham-operated groups(jlp-/--sham group and jlp+/+-sham group),jlp+/+-and jlp-/--unilateral ureteral obstruction (UUO)-operated groups (jlp/--UUO group and jlp+/+-UUO group).Mice were sacrificed at 7 days and 14 days after the operation respectively to evaluate the fibrosis by Masson staining.The expression of JLP in jlp +/+ renal tissue was assayed by immunohistochemistry staining,immunofluorescence and Western blotting.Immunohistochemical staining was used to detect the expression of α-smooth muscle actin (α-SMA),collagen Ⅰ (COL-Ⅰ),collagen Ⅲ (COL-Ⅲ) and transforming growth factor-β1 (TGF-β1) in sham and UUO groups.Besides,the α-SMA,COL-Ⅰ,COL-Ⅲ,TGF-β1,p-Smad2 and p-Smad3 protein levels were also analyzed by Western blotting in four groups.Results The expression of JLP was mainly demonstrated in the renal tubules of mice.A large amount of collagen deposition was observed in the renal interstitial area in jlp-/--UUO group compared to jlp+/+-UUO group.Similarly,the expression of α-SMA,COL-Ⅰ,COL-Ⅲ and TGF-β1 was significantly increased in the kidney cortices in jlp-/--UUO-operated groups.Meanwhile,Western blotting showed that the expression of α-SMA,COL-Ⅰ,COL-Ⅲ,and TGF-β1 protein was obviously higher in jlp-/--UUO group.Moreover,the expression of p-Smad2 and p-Smad3 protein was markedly higher in jlp-/--UUO group.Conclusion Scaffolding protein JLP is critical in preventing renal fibrosis through the inhibition of TGF-β1 expression and myo-fibroblast production.

Objective To investigate the expression of PTTG and VEGF-C in laryngeal carcinoma and the effect of angiogene-sis .Methods Immunohistochemistry was used to detect the expression of PTTG 、VEGF-C and LMVD in 60 cases of laryngeal car-cinoma and 32 cases of para-carcinoma tissue .D2 40 positive products was used to locate lymphatic endothelial cell cytoplasm and cell membrane ,and count lymphatic microvessel density (LMVD) .Results The expression of PTTG and VEGF-C in laryngeal car-cinoma was significantly higher than that in para-carcinoma tissue(P<0 .05) .In laryngeal carcinoma ,the expression of PTTG and VEGF-C were associated with differentiation ,lymph node metastasis and clinical stage(P<0 .05) ,but independent of clinical classi-fication ,smoking history ,tumor′size ,sex and gender(P>0 .05) .The expression of LMVD in laryngeal carcinoma was significantly higher than that in para-carcinoma tissue(P<0 .05) .The expression of LMVD was associated with lymph node metastasis (P<0 .05) ,but independent of differentiation ,clinical classification ,clinical stage smoking history ,tumor′size ,sex and gender ( P>0 .05) .A significantly positive relation was found between PTTG and VEGF-C(P<0 .05) .And there were positive relation between PTTG and LMVD、VEGF-C and LMVD(P<0 .05) .Conclusion PTTG and VEGF-C might play an important role in the carcino-genesis and development of laryngeal carcinoma .PTTG and VEGF-C could be a prognostic factor of colorectal cancer and a new tar-get of gene therapy .

Objective The aim is to observe the role and mechanism of estradiol ( E2 ) on the proliferation of rat hippocampal neural stem cells ( NSCs ) .Methods Twenty hippocampi from embryonic 17-day ( E17 ) SD rats were dissociated and plated into culture flasks with NSCs specific medium containing different concentrations of estradiol .The proliferation and the vitality of NSCs were detected by immunofluorescence against BrdU and MTT assay .The expression of estrogen receptors ( ERαand ERβ) was measured by immunofluorescence staining combined with Nestin double labeling . Results BrdU and MTT assay results showed that the cell number increased when the concentration of estradiol increased from 10 -10 to 10 -8 mol/L.The number of cell proliferation and the viability of cells were best at the concentration of 10 -8 mol/L compared to the other groups .However, when the estradiol concentration was increased from 10-8 to 10 -6 mol/L, the cell proliferative capacity declined gradually .Double immunofluorescence labeling showed that the two types of estrogen receptors ( ERαand ERβ) were expressed in the cultured hippocampal NSCs .Conclusion Estradiol promotes the proliferation of hippocampal NSCs in a certain concentration range , and ERαand ERβmay be involved in the estradiol-induced proliferation .