Objective To determine whether cultured neural stem cell expresses morphogen molecule sonic hedgehog′s functional receptor patched. Methods Cultured neural stem cell clones were subjected to RT\|PCR after passaged several times in vitro ,the amplification production was sequenced and labeled by digoxingemin and in situ hybridization technique was carried out to detect the cryosection of the neural stem cell clones. Results Most cells in the stem cell clones were positive for sonic hedgehog functional receptor patched,no significant difference was found among the positive cells and the center and peripheral of the stem cell clones.Conclusion\ The sonic hedgehog signal transduction may have important role in the proliferation and differentiation process of neural stem cell.\;[

Objective To investigate the influence of gene transfection antiangiogenesis on microvessel and relative cytokine of hypertrophic scar of rabbits' ear.Methods The hypertrophic scar of rabbtis' ear was reproduced.On the 10th day after epithelization,Ad-METH-1 was injected into tissue of scar.30 days later,the microvessel of scar-tissue was detected by microcirculation microscope.Meanwhile.H&E and immunohistochemical stains were performed.Then the results were analyzed.Results 30 days after Ad-METH-1 injection.in experimental groups,the microvascular count of scar tissue was 12.38±2.56,the percentage of VEGF positive cells was 17.64%,and the percentage of bFGF positive cell was 18.24%:while in the control groups,the microvascular count of scar tissue was 48.12±6.46.the percentage of VEGF positive cell was 31.34%.and the percentage of bFGF positive eell was 28.26%.Results revealed that the count of microvessel of scar tissue in the experimental groups was lower than that in the control groups,between which there was the difference in statistics(P＜0.01).and that the percentage of VEGF and bFGF positive cells of scar tissue in the expenmental groups were lower than that in the control groups.between which there was the difference in statistics(P＜0.05).Conclusion Ad-METH-1 has marked inhibitory effects on scar tissue hyperplasia of rabbits' ear,angiogenesis and expression of VEGF and bFGF.Using antiangiogenesis therapy at the early phase could inhibit the formation of hypertrophic scar.Gene transfection antiangiogenesis therapy could bid fair to become an effective method to prevent and treat hypertrophic scar.

Objective To investigate the mechanism of recombinant human bone morphogenetic protein-2(rhBMP-2)in repairing hematopoietic injury in mice irradiated with γ-ray.To prepare SRY gene probe and study the effect of rhBMP-2 in repairing hematopoietic injury in mice by in situ hybridization.Methods Twenty-two BALB/c female mice were randomly divided into the irradiated group and BMP treated group,respectively.Bone marrow cells of normal male mice were transplanted into 22 female mice post-irradiation to 8.5 Gy of 60 Co γ rays.The left femurs of the survived female mice were re-irradiated with 9 Gy 14 days later.Mice in BMP treated group were given rhBMP-2 20 mg/kg while those in control group were treated with 0.9% saline by intraperitoneal injection every day for 6 days.These mice were killed 14 days later and paraffin sections of femurs were made.The SRY gene was detected with in situ hybridization.Results There were more positive blots in the left femurs of the mice in irradiated group than those in BMP treated group(T=155.0,P＜0.05).The number of positive blots between the left femurs of the mice in irradiated group and the right femurs of the mice in two groups was not significantly different(T=92.0,78.5,P＞0.05).The number of positive blots in the left femurs of the mice in BMPtreated group was significantly less than those in the right femurs of the mice in two groups(T=155.0,55.0,P＜0.05).Conclusions No donor cell of male mice was detected in the left femurs of BMP treated group,suggesting that rhBMP-2 promoted the restoration of residuary bone marrow cells.Thus,rhBMP-2 promotes the proliferation or differentiation of residuary mesenchymal stem cells,improves hematopoietic microenvironment and accelerates the hematopoietic restoration.

AIM: To prepare Nano-Liposome encapsulated MAGE3/HSP70(NL M3H) and study its character and antitumor immunity in mouse. METHODS: NL M3H was prepared by the thin film-dispersion ultrasonic. The shape and size of NL M3H were detected by electron microscope. The encapsulation rate, drug-carrying capacity, stability and the releasing character were tested by Sephedex-G100 gel filtration. The mouse was immunized by NL M3H, and the antitumor immunity was detected by ELISPOT and LDH release assay. RESULTS: The mean size of NL M3H was lower than 100 nm. The encapsulation rate was 38％.The drug content was 0.038 g/L. NL M3H has good stability after stored in 4℃ for 6 months. The releasing profile showed that 74 percent of proteins was released during the first 24 hours in saline. The results of ELISPOT and LDH release assay showed that NL M3H generated tumor specific cytotoxic T lymphocyte(CTL)to damage tumor cel1. CONCLUSION: NL M3H has novel characters, it can generate specific CTL to kill tumor cell, and can be used as new kind of vaccine agsinst tumor.

Objective To investigate the expression of anti-apoptosis gene bag-1 and its relation to the differentiation of intrahepatic cholangiocarcinoma (ICC). Methods Immunohistochemical techniques were adopted to study the expression of bag-1 in ICC tissue ( n=48) and para-hepatocarcinoma bile duct ( control group, n=25). Results Expression of bag-1 in the ICC group was significantly higher than that in the control group. In the ICC group (P

Human nerve growth factor (NGF) is a nerve cell growth regulation factor, which can provide nutrition for the neurons and promote the neurites outgrowth. In order to produce large-scale recombinant human nerve growth factor (rh-beta-NGF), we constructed a plasmid vector, which can stably express the rh-beta-NGF in the HEK293 cell lines. First, the plasmid of pCMV-beta-NGF-IRES-dhfr was constructed and transformed into HEK293 cells. Then MTX pressurized filter and limiting dilution methods were used to obtain monoclonal HEK293 cell lines. After stepwise reducing serum in culture media, the cells eventually adapted to serum-free medium and secreted rh-beta-NGF. SDS-PAGE analysis revealed that the expression product owned a molecular weight of about 13 kDa and a purity of more than 50%. The peptide mapping sequencing analysis demonstrated the sequences of rh-beta-NGF matched with the theoretical ones. Later we purified this protein by ion exchange and molecular sieve chromatograph. Finally, our experimental results exhibited that the recombinant cell lines can stably express rh-beta-NGF with a high efficiency of more than 20 pg/cell x day. In addition, this protein could successfully induce differentiation of PC12 cells. In summary, our recombinant HEK293 cells can express bio-active rh-beta-NGF with great efficiency and stability, which supply a valid basis to large-scale production of rh-beta-NGF.
Cell Differentiation ; Genetic Vectors ; HEK293 Cells ; Humans ; Nerve Growth Factor ; biosynthesis ; Plasmids ; Recombinant Proteins ; biosynthesis

An improved and practical synthesis of racemic 11-demethylcalanolide A [(±)-1] was developed. This improved process involved Pechmann reaction on phloroglucinol with ethyl butyrylacetate to give 5, 7,-dihydroxy-4-n-propylcoumarin(3). Poly phosphoric acid (PPA) catalyzed acylation of compound(3) with crotonic acid, then intramolecular cyclization was achieved simultaneously in one step to afford the key intermediate chromanone(4). A microwave assisted synthetic method preparing chromene(6) using chromenynation of chromanone(4) with 1, 1-diethoxy-methyl-2-butene was conducted. Luche reduction of chromene(6) using NaBH<,4> with CeCl3·7H2O preferably gave (±)-1. The overall yield of this four step synthesis of (±)-1 was around 32% increasing one fold more than that of the previous method. An in vitro investigation showed that (±)-1 exhibited inhibitory activities against both wild-type and drug-resistant HIV-1 in HIV-1 RT and cell culture assay, and significant synergistic effects in combination with AZT, T-20, and indinavir. Its LD50 of acute toxicity in mice by intragastric administration and by intraperitoneal injection were 735.65mg·kg-1 and 525.10mg·kg-1, respectively. The C<,max> and AUC<,0-∞> were 0.54μg·mL-1 and 1.08(μg·mL-1)·h, respectively. The dynamics study of the inhibition of mice sera on HIV-1 RT showed that mice treated with 100mg·kg-1 (±)-1 once intraperitoneally were similar to that of 5mg·kg-1 of known clinical effective anti-HIV-1 drug neverapine. The results suggested that further investigation of the anti-HIV candidate (±)-1 was warranted.

Objective To understand the pathway of iodine intaking among Tibetan, and provide basic data for prevention and control of iodine deficiency disorders (IDD). Methods Through the method of random sampling, the boarding and day student aged 8 - 10 and women of childbearing age were conducted dietary survey to understand the condition of food intaking via the 24 h review method in 2015. Samples of urine, drinking water, dried beef, milk, Qula and fried noodles were collected and tested iodine level. Results Due to taking iodized salt three times a day with meals, the median of urinary iodine among 492 investigated boarding students was 179.2 μg/L;differently, the median of urinary iodine among 298 day students in this investigation was 79.6 μg/L who taking iodized salt only at lunch at school;and in the study, the median of urinary iodine among 158 women of childbearing age who took iodine-free salt daily was 33.7 μg/L. The iodine contents in 51 drinking water samples, 66 dried beef samples, 48 milk samples, 20 Qula samples and 37 fried noodle samples were quantified respectively, and the average iodine contents of each food were 0.8 μg/L in drinking water, 59.1 μg/kg in dried beef, 61.5 μg/kg in milk, 226.4 μg/kg in Qula and 17.0 μg/kg in fried noodles. The acceptable daily intake (ADI) of iodine of the boarding and day students aged from 8 to 10 and women of child bearing age were 234.0, 126.4 and 76.7 μg/d, respectively, among which the ADI of iodine with iodized salt were 208.0, 78.0 and 0.0 μg/d. Conclusion Consuming iodized salt is a main method to get iodine among Tibetans in Nangqian County, so that it is significant to carried out this measure for a long time for free to let them have iodized salt every day instead of iodine-free one.

Objective:To investigate the dietary iodine intake of people in different areas of Qinghai Province, and to provide the basis for scientific iodine supplementation and continuous elimination of iodine deficiency hazards.Methods:From 2018 to 2019, according to administrative division, natural geographical regions, population distribution and economic development level of Qinghai Province, a total of 14 survey sites were selected. One village was selected from each survey site, and 20 households were selected from each village, the salt samples and 24 h urine samples of all family members were collected to detect salt iodine and urinary iodine. One drinking water sample was collected at the five directions of east, west, south, north and middle of each village to detect water iodine. Salt iodine was detected by direct titration, urinary iodine and water iodine were detected by arsenic-cerium catalytic spectrophotometry. At the same time, the 3-day weighing method was used to investigate the diet, the daily dietary iodine intake per capita (the result was expressed as average) and the proportion of dietary iodine in urinary iodine were calculated, the daily dietary iodine intake per capita of different production modes (agricultural region and pastoral region), different geographical environment (Hehuang Valley, Qaidam Basin, Qilian Mountain and Qingnan Plateau), different nationalities (Han, Tibetan, Hui, Mongolian, Tu, Salar) and different economic levels (< 8 000, 8 000 -, 10 000 -, ≥12 000 Yuan) were compared.Results:A total of 999 people from 280 families were surveyed, including 511 males and 488 females. The median water iodine of each survey site was less than 10 μg/L, all of which were environmentally iodine-deficient areas. A total of 280 salt samples were collected, the median salt iodine was 26.0 mg/kg, and the consumption rate of qualified iodized salt was 100% (280/280). A total of 999 urine samples were tested, and the median urinary iodine of people was 192.5 μg/L, which was at an appropriate level of iodine. There was no statistically significant difference ( t =-1.599, P > 0.05) in the daily dietary iodine intake per capita (28.53, 33.44 μg) of people in agricultural region ( n = 643) and pastoral region ( n = 356). The daily dietary iodine intake per capita (25.38, 33.30, 32.98, 34.79 μg) of people in Hehuang Valley ( n = 448), Qaidam Basin ( n = 125), Qilian Mountain ( n = 157), and Qingnan Plateau ( n = 269) were compared, the difference was statistically significant ( F = 2.883, P < 0.05); among them, the daily dietary iodine intake per capita in Hehuang Valley was lower than that in Qingnan Plateau ( P < 0.05). The daily dietary iodine intake per capita of different nationalities were compared, the difference was statistically significant ( F = 3.647, P < 0.05), Salar ( n = 68) and Tibetan ( n = 239) were higher (37.21 and 32.21 μg). The daily dietary iodine intake per capita (38.97, 17.01, 30.86, 33.14 μg) of annual per capita disposable income < 8 000 ( n = 194), 8 000-( n = 221), 10 000-( n = 302), ≥12 000 Yuan ( n = 282) were compared, the difference was statistically significant ( F = 9.407, P < 0.05). The proportions of dietary iodine in urinary iodine of various population ranged from 5.35% to 15.54%. Conclusions:The iodine nutrition of people in Qinghai Province is suitable, the dietary iodine intake of people is closely related to geographical environment, nationality and economic level. But the proportion of dietary iodine in urinary iodine is relatively low, the consumption of iodized salt is still the main way for people to intake iodine, and it is also the main measure to continuously eliminate the harm of iodine deficiency in Qinghai Province.

Objective:To verify the revised method of cerium sulfate catalytic spectrophotometry for iodide index of "Standard Examination Methods for Drinking Water-Nonmetal Parameters" (GB/T 5750.5-2006).Methods:From July to September 2019, the Laboratory of Department for Endemic Disease Prevention and Control of Qinghai Institute for Disease Prevention and Control verified the revised method (determination of iodide in drinking water by cerium sulfate catalytic spectrophotometry) of cerium sulfate catalytic spectrophotometry (hereinafter referred to as original method) in "Standard Examination Methods for Drinking Water-Nonmetal Parameters" (GB/T 5750.5-2006). The revised method was verified according to the requirements of "Standard Examination Methods for Drinking Water-Water Analysis Quality Control" (GB/T 5750.3-2006), including standard curve, detection limit, precision, accuracy and actual sample determination.Results:The linear range of the revised method was 0 - 20.0 μg/L, the correlation coefficient was - 0.999 4 - 0.999 8, and the detection limit was 0.231 μg/L. The relative standard deviation ( RSD) of low, medium and high iodine water samples of 6 times detection ranged from 1.4% to 9.6%, and the recoveries of low and medium water samples ranged from 89.0% to 108.0%. The detection results of national first-class reference materials for iodine composition analysis in water were within the range of standard value ± uncertainty. There was no significant difference in the test of results of 12 tap water samples between the revised method and the original standard method ( t = - 0.075, P > 0.05). Conclusion:The revised method has a good linear relationship of standard curve, high precision and accuracy, and good reproducibility, is simple and easy to operate, and is suitable for promotion and application.