Objective To establish immortalized neural progenitor cell strain and provide stable cell resource for cell-transplantation and gene therapies. Methods Plasmid pCMVSV40T/PUR containing the simian virus 40 large T antigen gene (SV40Tag) were transfected into the primary cultured neural progenitor cells (NPCs) of newborn rat using lipofectin transfection method. Colonies were isolated by puromycin selection and expanded by many passages. Anti-nestin antibodies were used to identify the cultured cells. The specific molecular marker of neurons and astrocytes were detected using immunocytochemistry method to investigate the capability of differentiation of the transfected cells. The expression of SV40Tag in expanded cell lines was identified by RT-PCR, Southern blot and immunocytochemistry method.Results One anti-puromycin cell clone was obtained, which was microtubule-associated protein-2 (MAP-2) positive cells with the capability of proliferation and could differentiate into MAP-2 or glial fibrillary acidic protein positive cells. The existence of SV40Tag cDNA and the expression of mRNA and protein of SV40Tag were confirmed in transfected cells. The transfected cells were expanded to immortalized cell strain maintained for more than 50 passages, named as immortalized neural progenitor cell (INPC) . INPCs were elliptical or triangular cells with two or three short axons. The population doubling time of INPC was (22.9?2.7)h, subculture, freezing and recovering had no effect on cellular shape and proliferation of INPC. Conclusion Immortalized neural progenitor cell strain was established successfully. It may provide stable cell resource for the basic researches and cell-transplantation therapies with NPC.

To construct an immortalized rat astrocyte strain genetically modified by rat preprogalanin gene (IAST/GAL) and detect its galanin (GAL) expression and secretion, a cDNA fragment of rat GAL in plasmid of pBS KS(+)-GAL was inserted into eukaryotic expression vector pcDNA3.1 (+) by DNA recombinant technology, then the restriction enzyme digestion and DNA sequencing were carried out to evaluate the recombinant. The pcDNA3.1 (+)-GAL and pcDNA3.1 (+) construct were transfected into immortalized rat astrocyte strain (IAST) by lipofectamine and the population of cells which stably integrated the construct was selected with 600 microg/mL G418. Individual clones were screened and expanded into clonal cell strains. Detection of Neo gene was used to validate the success of the transfection. Immunocytochemical staining, RT-PCR and radioimmunoassay were used to detect the expression and secretion level of GAL. The recombinant had been successfully constructed by restriction enzyme digestion and DNA sequencing. Detection of Neo gene showed that the pcDNA3.1 (+)-GAL and pcDNA3.1 (+) have been successfully transfected into IAST. After selection by using G418, IAST/GAL and IAST/Neo cell strains were obtained. IAST/GAL, IAST/Neo and IAST were immunostained positively for GAL, but the GAL average optical density of IAST/GAL was significantly higher than that of IAST/Neo and IAST (P< 0.01). The level of GAL mRNA expression and the supernatant concentration of GAL in cultured IAST/GAL were significantly higher than those of IAST and IAST/Neo (P<0.01), but no significant differences were found between the IAST and IAST/Neo (P>0.05). It was concluded that IAST/GAL strain was constructed successfully and it might provide a basis for the further study of pain therapy.
Astrocytes/cytology ; Astrocytes/*metabolism ; Cell Line, Transformed ; Cells, Cultured ; Galanin/*biosynthesis ; Galanin/genetics ; Genetic Vectors ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; Recombinant Proteins/biosynthesis ; Recombinant Proteins/genetics ; Transfection

Objective To investigate the mRNA and protein expression of E3 ubiquitin ligase Iduna in non-small-cell lung cancer tissue and para-neoplastic lung tissue,and the correlation of the Iduna expression with clinicopathological factors and prognosis.Methods The expression levels of the Iduna mRNA and protein in non-small-cell lung cancer tissue and para-neoplastic lung tissue were determined by reverse transcriptase-polymerase chain reaction (RT-PCR),Western-Blot and immunohistochemistry respectively,and the correlation of the Iduna expression with clinicopathological factors and prognosis was analyzed.Results RT-PCR and Western-Blot showed the expression levels of the Iduna mRNA and protein in non-small-cell lung cancer tissue (0.468 ± 0.086 and 2.554 ± 0.544) were significantly higher than those in para-neoplastic lung tissue (0.203 ± 0.070 and 1.570 ± 0.316),there were statistical differences (P ＜ 0.05).Immunohistochemistry results showed that Iduna was negative expression in the alveolar epithelium cells,negative or weak positive expression in normal bronchial and positive expression in different degrees in the non-small-cell lung cancer tissue.Iduna high expression rate was negative correlation with tumor differentiation (P =0.002),Iduna high expression rate was positive correlation with large tumor size (P =0.044),TNM staging (P=0.015) and lymph node metastasis (P=0.009).Iduna high expression of I stage non-small-cell lung cancer patients was correlated with poor post-operative survival (P =0.016).Conclusions High expression of Iduna may be related to the process of invasion and metastasis of nonsmall-cell lung cancer.It is possible that Iduna serve as potential markers for predicting prognosis in nonsmall-cell lung cancer.

Objective To construct lentivirus vector expressing antigene RNA and ferritin gene.Methods Intermediate plasmid pGC-FU-HF was constructed by transfecting lentivirus vector pGC-FU with heavy chain ferritin subunit gene.The target plasmid pGC-agRNA-HF was subsequently constructed by transfecting the intermediate plasmid with β-arrestin 2 antigene RNA.The NG108-15 cells were transfected with the target plasmid.The titre of lentivirus vector was measured by RT-PCR.The expression of antigene RNA and ferritin gene was determined by Western blot and RT-PCR.Results Lentivirus vector was successfully transfected with antigene RNA and ferritin gene.The titre of lentivirus vector was 2.00 × 109 TU/ml.The expression of β-arrestin2 protein was down-regulated and the expression of ferritin protein up-regulated in the NG108-15 cells after being transfected with the lentivirus vector.Conclusion Lentivirus vector expressing antigene RNA and ferritin gene has been successfully constructed.

Descending nociceptive modulation from the supraspinal structures plays an important role in cancer-induced bone pain (CIBP). Rostral ventromedial medulla (RVM) is a critical component of descending nociceptive facilitation circuitry, but so far the mechanisms are poorly known. In this study, we investigated the role of RVM glial activation in the descending nociceptive facilitation circuitry in a CIBP rat model. CIBP rats showed significant activation of microglia and astrocytes, and also up-regulation of phosphorylated p38 mitogen-activated protein kinase (p38 MAPK) and pro-inflammatory mediators released by glial cells (IL-1β, IL-6, TNF-α and brain-derived neurotrophic factor) in the RVM. Stereotaxic microinjection of the glial inhibitors (minocycline and fluorocitrate) into CIBP rats' RVM could reverse the glial activation and significantly attenuate mechanical allodynia in a time-dependent manner. RVM microinjection of p38 MAPK inhibitor (SB203580) abolished the activation of microglia, reversed the associated up-regulation of pro-inflammatory mediators and significantly attenuated mechanical allodynia. Taken together, these results suggest that RVM glial activation is involved in the pathogenesis of CIBP. RVM microglial p38 MAPK signaling pathway is activated and leads to the release of downstream pro-inflammatory mediators, which contribute to the descending facilitation of CIBP.

OBJECTIVEImproving the traditional fermentation technology by some unidentified strains into the modern pure inoculation fermentation technology. For selecting better fermentation strains, the quality of various Shenqu fermentated with different pure inoculation be compared.

METHODIsolating the strains from self-fermented Shenqu, then with other 2 certain strains, to conduct the pure inoculation fermentation. Then compare the quality of them according to the difference in the chemical composition, activity of enzyme and the impact to the digestive function in mice of the finished Shenqu.

RESULTSFour strains were isolated. One of them, a mucor genus of mold is better than others.

CONCLUSIONModern pure inoculation fermentation can ensure the quality and the medication safety of Shenqu. New fermentation technology is feasible.

Animals ; Bacillus subtilis ; growth & development ; metabolism ; Drugs, Chinese Herbal ; metabolism ; pharmacology ; Female ; Fermentation ; physiology ; Food Microbiology ; Male ; Mice ; Random Allocation ; Stomach ; drug effects ; metabolism

BACKGROUND: Antigene RNAs (agRNAs) could be a useful tool to downregulate beta arrestin 2 (Arrb2) gene expressions, and realize gene knock-out effect in cell levels. OBJECTIVE: To observe the effects of agRNAs on DAMGO-induced mu-opioid receptor (MOR) desensitization in C17.2 cells by using agRNAs complementary to transcription start sites of beta arrestin 2 (Arrb2) to downregulate the gene expression in mouse C17.2 cells. METHODS: Mouse neural stem cells C17.2 was cultured in Dulbecco's minimal essential medium containing 10% fetal bovine serum, 10 mg/L penicillin and 10 mg/L ampicillin with 5% CO_2 at 37 ℃. The cells were passaged every 5 to 6 days after digestion with 0.25% trypsin when cells were 80% confluent. The expression of MOR on mouse C17.2 cells was detected by RT-PCR and immunocytochemical method. AgRNAs which could silence the expression of Arrb2 was transfected into C17.2 cells with Lipofectamine. The expression of green fluorescent protein gene was observed by fluorescence microscopy 24 hours after transfection. [~(35)S]GTPγS binding was assessed by autoradiography to examine the ability of the MOR to couple to G proteins on stimulation with selective agonist DAMGO.RESULTS AND CONCLUSION: The expression of MOR on C17.2 cells was confirmed by RT-PCR. The receptors were expressed on cell membrane and in plasma determined by immnocytochemistry. The expression of green fluorescent protein gene could be observed in C17.2 cells transfected with plasmid pGPU6/GFP/Arrb9 using fluorescent microsocpe. The results of [~(35)S]GTPγS binding showed that the stimulation ratio in cells with and without DAMGO stimulation or transfected with agRNAs were (113±14)%, (253±17)% and (239±15)% respectively. This indicated that agRNAs could downregulate the expression of beta-arrestin 2 and inhibit the desensitization of MOR.

Objective: To detect the systolic and diastolic velocity in patients with idiopathic premature ventricular beat(PVB)by pulsed wave-tissue Doppler imaging (PW-TDI) techniques.Methods: There were two groups involved in this study. PVB group,n=30,patients with idiopathic PVB,and Control group,n=30,of healthy subjects. The changes of cardiac function were measured and compared between two groups by the index of myocardial motion velocity using PW-TDI techniques.Results: A total of 7 sites were studied,there were no significant differences in systolic peak velocities (Sm),time velocity integral (TVI),peak velocity of the early relaxation wave (Em),peak velocity of the later relaxation wave (Am) and Em/Am ratio (Em/Am) between the Control group and PVB group in the normal sinus beat (P＞0.05).Compared with the normal sinus beat,Sm,TVI,Em and Em/Am were significantly lower in PVB (P<0.01),while Sm,TVI,Em in the post-PVB sinus beat were significantly higher than that in normal sinus beat (P<0.01). However,there were no significant differences of Am in each site among the normal sinus beat,PVB and the post-PVB sinus beat. Conclusion: PVB and PVB-induced compensational interval could significantly influence the systolic and diastolic function of the ventricle.

Objective To evaluate the feasibility of using comprehensive geriatric assessment (CGA) in estimating if standard dose treatment is fit for the elderly patients with diffuse large B cell lymphoma.Methods.Comprehensive geriatric assessments including three assessments of activity of daily living,instrumental activity of daily living and comorbidity scoring according to Cumulative Illness Rating Score for Geriatrics were adopted to assess if standard dose treatment is fit for the elderly patients in our prospective study.Thirty seven patients with diffuse large B cell lymphoma,aged ＞70 years were enrolled in the study,and grouped into fit,unfit and frail groups according to comprehensive geriatric assessment scoring and their age.The treatment protocolswere not determined by comprehensive geriatric assessment scores,but by clinical judgments made by clinicians based on their clinical experience and disease features.The clinically effective response and overall survival (OS) were analyzed in the three groups.Results According to CGA scores,patients were grouped into fit [21 cases (56.8％)],unfit [7 (18.9％)] and frail [9 (24.3％)].37 cases received 213 courses of treatment at average 5.76 courses per case.The overall response (complete / partial remission) rates were [85.7％(18/21) vs.28.6％ (2/7) vs.44.4％ (4/9),x2=9.69,P=0.008] and median survival times were (44 months vs.10 months vs.9 months;x2 =7.03,P=0.03) among fit,unfit and frail groups with statistically significant differences.Total effective rate (achieving all clinical targets) in fit group of 21 cases were 100 ％ (12/12)with receiving standard dose therapy,and 66.7％ of(6/9)with low dose therapy(P=0.06).Overall response rate(total/partial remission) [85.7％(18/21) vs.28.6％(2/7) vs.44.4％(4/9),x2=9.69,P=0.008] and median survival (44 months vs.10 months vs.9 months;x2 =7.03,P=0.03) amongfit,unfit and frail groups.In fit group,the two-year overall survival was higher in patients receiving standard dose treatment than receivingpalliativetreatment,with statistical significance [83.3 ％ (10/12) vs.33.3 ％ (3/9),P =0.032],without significant hematologic toxicity observed between the subgroups.Conclusions Comprehensive geriatric assessment can identify if elderly patients diffuse large B cell lymphoma can acquire a satisfactory curative effect from a standard dose treatment ofimmunochemotherapy.

Objective To investigate the change in 5-hydroxytryptomine (5-HT) content in spinal dorsal horn in a rat model of tibial bone cancer pain (BCP). Methods Sixty female SD rats weighing 160-180 g were randomly divided into 3 groups ( n = 20 each): control group (group C), sham operation group (group S) and BCP group. BCP was induced by intra-tibial inoculation of 10 μl Walker 256 breast cancer cell suspension in group BCP, while group S received intra-tibial inoculation of 10 μl D-hank solution. Paw withdrawal threshold to mechanical stimulation with yon Frey filaments (MWT) was measured 1 d before (baseline) and at 3, 5, 7, 9, 11,14, 16, 18 and 21 d after breast cancer cell inoculation. At 1 d before and 7, 14 and 21 d after breast cancer cell inoculation, four animals in each group were sacrificed after measurement of MWT. Their lumber segments of the spinal cord were removed for assay of 5-HT content in spinal dorsal horn using HPLC with fluorescence detector.HE staining was used to detect the damage to the tibia. Correlation between the 5-HT content and MWT was analyzed. Results MWT was significantly decreased after breast cancer cell inoculation in group BCP ( P ＜ 0.05).Microscopic examination showed serious bone destruction of tibia at the injection site in group BCP, while no bone destruction was found in groups C and S. 5-HT content in spinal dorsal horn was significantly higher in group BCP than in groups C and S (P ＜ 0.05). There was strong negative linear correlation between 5-HT content in spinal dorsal horn and MWT ( r = - 0.973, P ＜ 0.05 ). Conclusion The 5- HT content in spinal dorsal horn is significantly increased in rats with tibial BCP and is involved in the development of BCP.