To evaluate whether in vitro and in vivo transfer of E. Coli cytosine deaminase gene will confer sensitivity of a solid tumor to prodrug 5-fluorocytosine(5FC), we used an adenovirus vector(AdexCMV. CD) carrying the cytosine deaminase gene driven by the CMV promoter, infected SMMC-7721 or HepG2 cells hepatocellular carcinoma cells in vitro, and found AdexCMV. CD vector could effectively suppressed the growth of SMMC-7721 and HepG2 cells. When the two cells were infected with AdexAFP. CD vector in which the CD gene was driven by the AFP gene 5'-flanking region, only HepG2 cells were conferred sensitivity to 5FC. (Infection with AdexCMV.CD, when as few as 20% of cells transfected the CD gene, SMMC-7721 cells were associated with a bystander effect when combined with 5FC in cell mixing studies.) Consistent with these in vitro observations, AdexCMV. CD was directly injected into established subcutaneous SMMC-7721 tumors in nude mice receiving 5FC,there was a 60% reduction in tumor size at day 8, 70% reduction at day 24. Our results suggested that adenovirus-mediated tumor-specific gene transfer of CD gene and concomitant administration of 5 FC may have potential as a strategy for local control of tumor growth.

Interleukin-17 (IL-17) is a newly found cytokine secreted by activated T lymphocytes. From preliminary study, IL-17 was found to play a role in the relationship between T lymphocytes and hematopoiesis, but many of its characteristics remain unknown up to now. To investigate its biological functions and related mechanisms, we cloned the complete coding region of human IL-17(hIL-17) from activated human peripheral blood mononuclear cells (PBMC) by RT-PCR and constructed an integrative vector pLCM182-hIL-17 for cloning, expressing and sequencing the hIL-17 gene. By DNA sequencing, the cloned hIL-17 is identical to the reptorted, and with temperature inducing, the hEL-17 was highly expressed in E. coli up to 30% of bacterial protein. The expressed hIL-17 protein could stimulate primary human fibrob-lasts to secrete IL-6 and GM-CSF after initial purification. This results indicate that the expressed hIL-17 has biological activity.

To further investigate whether the suicide gene therapy affect the immune system, CT26 colon adenocarcinoma of BALB/c mice was adapted as experimental tumor model. The growth of CT26 tumor was suppressed by the adenovirus-mediated CD/5FC system significantly both in vitro and in vivo. But the tumors seemed very difficult to be cured and developed again soon after the end of treatment. After adenovirus-mediated CD/5FC gene therapy, there were about 40%of treated mice were cured from the tumor burden, survived longer and resisted further CT26 cell challenge. These mice also showed improvement of splenic CTL cytotoxicity. Costimulatory molecule B7-2, dendritic cell marker NLDC-145, MHC-I, and F4/80 were present in tumor mass in mice. These data suggested that in vivo suicide gene therapy could induce specific antitumor immunity, but this effect is not strong enough to eradicate established tumors. Further study for more efficient induction of antitumor immunity in CD/5FC gene therapy is warranted.

Liver ferritin of Sphyrna zygaena(SZLF) with purity of mass spectrum was prepared in batch. Under) the condition of acidifying medium at pH 1.0, PAGE showed that SZLF subunits treated for 20 min began) to dissociate. A whole process of subunit dissociation and recombination was monitored by transmission electron microscopy(TEM). In addition, the changes of size of both protein shell and iron core were also determined) by TEM directly. It was found that in the acid dissociation process of SZLF subunits, the size of iron) core and protein shell showed the same trend of change, which might be related to not only the iron release) of inner iron core but the dissociation and unfolding of the protein shell. The passway of SZLF recombination is a fast step, which is a conversion process from incompact moltenglobule to compact ferritin. Under the assistant of matrix acidity pH 3.0 and laser, SZLF mixed with horse spleen ferritin(HSF) still has capacity to release) its subunits to form subunit ions for mass analysis by a MALDI-TOF mass spectrometer, which indicates that the interaction intensity between the subunits was weaken but they were not unfolded under this pH condition. TEM technology can be applied in studying both dissociation and recombination in ferritin subunits.

Objective To investigate the effect of catch-up growth on insulin resistance(IR) through analysis of biochemical and metabolic indices in premature infants.Methods There were 126 infants admitted in the Department of Neonatology,the Affiliated Hospital of Guiyang Medical College from December 2010 to December 2013 [factors which might affect the secretion of insulin(INS),C peptide and insulin-like growth factor-1 (IGF-1) were excluded].According to gestational age and birth weight,babies were divided into small for gestational age (SGA) group and appropriate for gestational age (AGA) group.And according to the age on follow-up,babies were divided into 1-to-6-month-old group,7-to-12-month-old group and ≥ 1-to-3 year-old group.All cases had 6 mL peripheral venous blood sampled in the early morning during fasting in the first week,and 6,12,24,36 months after birth.They were tested for levels of INS,C peptide,IGF-1,triglyceride (TC),total cholesterol (TG),high density lipoprotein cholesterol(HDL-C),low density lipoprotein cholesterol(LDL-C),fasting blood-glucose (FBG),albumin,prealbumin and creatinine respectively.At the same time,the physical parameters were measured,including weight,body length,and calculated insulin resistance index (IRI),Ponderal index,weight standard deviation score (SDS),and length SDS.Results (1) Catch-up growth after premature birth occupied 65.6％ (63/96 cases),whereas no catch-up growth occupied 34.4％ (33/96 cases) of study snbjects,and among them catch-up growth of 37 cases was better (8 cases of SGA,29 cases of AGA),26 cases showing catch-up growth(7 cases of SGA,19 cases of AGA),33 cases without catch-up growth(11 cases of SGA,22 cases of AGA).No statistical significance was found in the distribution of catch-up growth between SGA group and AGA group(P ＞ 0.05).(2) The LgIRI,LgINS of group with good catch-up growth was significantly lower than the group with no catch-up growth group (F =3.55,3.47) in infancy,but the level of IGF-1 and prealbumin was higher than that of no catch-up growth group (F =3.55,4.94),the difference had statistical significance (P ＜ 0.05) ; the better catch-up growth was associated with higher IGF-1 and prealbumin,but with lower LgIRI.(3) The risk factors for insulin resistance were SGA (OR =7.904,P =0.001),low birth weight ＜ 1 500 g (OR=8.737,P=0.019),and no catch-up growth (OR=11.706,P=0.000).Conclusions The better catch-up growth in infancy is associated with higher IGF-1 and prealbumin,but lower IR.The risk factors of IR include SGA,low birth weight and no catch-up growth,and the last being the major factor.

The hG -CSF cDNA was cloned by RT-PCR and confirmed by sequencing, which contains the full length of hG-CSF encoding region and parts of 5 '、3' non - coding region. Then the hG - CSF retroviral vector pLGSN was constructed by orientationally inserting the hG-CSF cDNA into the EcoRI/XhoI cloning sites of pLXSN vector. Packaged with CRE and CRIP packaging cell lines which are considered to be unlikely to produce helper viruses, the final pLGSN retrovirion titer reached 1. 1 ?106 CFU/ml. During constitutive passaging, the CRIP - LGSN cell clone produced relatively stable tilers of pLGSN retrovirion, ranging from 6. 8?105CFU/ml to 1. 1?106CFU/ml. By infecting the murine fibroblast cell line NIH3T3 with pLGSN retrovirion, a cell clone designated as NIH3T3 -G -CSF was obstained, secreting 168U/ml G-CSF . The integration and expression of hG-CSF gene in this cell clone were confirmed by Southern and Northern blotting analyses. Western blotting has also detected specifically the hG-CSF protein in the condensed supernalants from NIH3T3-G-CSF cells . After packaging the hG-CSF-secreting fibroblasts with collagen and implanting them into synergenic mice peritoneally , we detected a certain levels of G-CSF in the sera of mice, which suggested the implanted NIH3T3-G-CSF fibroblast cells could constitutively express and release hG-CSF in vivo. Our data showed the constructed hG-CSF retroviral vector could be used to further investigate the fibroblasl-mediated hG-CSF gene therapy .

Objective:To observe the clinical efficacy of ultrasound cycloplasty (UCP) in the treatment of uncontrolled intraocular pressure (IOP) after glaucoma surgery.Methods:An observational case series study was carried out.Twenty-eight consecutive patients (28 eyes) with uncontrolled IOP after glaucoma surgery who received UCP treatment in The First Affiliated Hospital of Zhengzhou University from July 2018 to October 2019 were enrolled.The IOP of these patients was ≥21 mmHg (1 mmHg=0.133 kPa) under the maximum tolerated dose.According to preoperative IOP and visual acuity, the patients were divided into 8-sector group (17 eyes) and 10-sector group (11 eyes). The duration of UCP operation, preoperative and postoperative 1-day, 1-week, 2-week, 1-month and 3-month IOP and BCVA, the types of drugs for lowering IOP preoperatively and postoperatively, preoperative and postoperative 3-month ocular pain grading and corneal endothelial cell counts, and adverse reactions during the operation and after surgery were recorded.This study adhered to the Declaration of Helsinki.The study protocol was approved by an Ethics Committee of The First Affiliated Hospital of Zhengzhou University (No.2020-KY-154). Written informed consent was obtained from each subject prior to any medical examination.Results:The duration of UCP operation was 3 to 7 minutes, with an average of (4.30±1.26) minutes.The IOP at 1 day, 1 week, 2 weeks, 1 month and 3 months after operation was (32.96±10.49), (25.89±7.25), (24.50±6.23), (24.07±6.59), (24.32±6.52)mmHg, respectively, which were significantly lower than (45.82±8.81) mmHg before operation (all at P<0.05). There was no significant difference in IOP between the 8-sector group and 10-sector group ( Fgroup=1.271, P=0.270), but there was a significant difference in IOP between the two groups before and after operation ( Ftime=54.388, P<0.01), and the postoperative IOP at various time points in the two groups were lower than the preoperative IOP, showing statistical significances (all at P<0.05). There was no significant difference in BCVA before and after surgery ( F=2.562, P=0.075). There was a statistically significant difference in BCVA between the 8-sector group and 10-sector group ( Fgroup=12.602, P=0.001), but no statistically significant difference was found in BCVA between the two groups before and after surgery ( Ftime=1.701, P=0.139), and the BCVA in the 8-sector group was better than the 10-sector group at various time points (all at P<0.05). The types of IOP lowering drugs used in the 8-sector group and 10-sector group were 3 (2, 3) and 3 (2, 4) before operation respectively, and 0 (0, 1) and 0 (0, 0) at 3 months after operation respectively.The preoperative ocular pain grade was 2 (2, 2), and the postoperative 3-month ocular pain grade was reduced to 1 (0, 1), and the difference was statistically significant ( Z=-4.824, P<0.05). The postoperative 3-month pain grading in the 8-sector and 10-sector groups were significantly lower than the preoperative pain grading ( Z=-3.739, -3.127; both at P<0.05). The corneal endothelial cell count was significantly decreased from (1 967.15±186.06) cells/mm 2 before operation to (1 861.08±206.63) cells/mm 2 at 3 months after operation ( t=2.781, P=0.017). No serious complications occured during the operation.Postoperative adverse reactions included chemosis and bulbar hyperemia, corneal edema, headache, ocular pain, anterior chamber inflammation, etc.Serious complications such as low IOP, macular edema, vision loss or eyeball atrophy were not observed. Conclusions:UCP has no surgical incision.Treatment of both 8 sectors and 10 sectors can effectively reduce IOP, reduce the types of IOP lowering drugs, and relieve ocular pain in patients with uncontrolled IOP after glaucoma surgery with few intraoperative and postoperative adverse reactions.

Objective To evaluate the efficacy of hypothermia therapy in neonates with hypoxic-ischemic encephalopathy(HIE)through the analysis of amplitude-integrated electroencephalography.Methods A retrospective analysis was conducted on 59 neonates with moderate to severe HIE.They were divided into observation group(n=31,hypothermia therapy)and control group(n=28,conventional therapy)according to different treatment protocols.Chi-square test,independent sample t-test,and one-factor Mann-Whitney U test were used for intergroup difference analysis.Results Significant differences between two groups were observed in lower boundary amplitude after 24 h of treatment,sleep-awake cycle after 72 h of treatment,and total scores after 72 h of treatment(P<0.05).After 48 and 72 h of treatment,the neonates in hypothermia therapy group had obviously lower neuro-specific enolase level than those in control group(P<0.05).Conclusion Early application of hypothermia therapy can significantly improve cerebral function in neonates with HIE and lower the neuro-specific enolase level.

Objective: To explore an optimal method for granulocyte cell production from umbilical cord blood mononuclear cells. Methods: Erythrocytes were precipitated by hydroxyethyl starch. Mononuclear cells were isolated through Ficoll density gradient centrifugation. Different media, additives and cultivation model were chosen for granulocyte induction. Cell morphology was observed by microscopy, and cell phenotype was detected by flow cytometry. The CD18 expression of granulocytes was tested by immunofluorescence assay, and phagocytosis test was executed as well. Results: Compared to fetal bovine serum (FBS) treatment group, cell viability, counts and differentiation rate of granulocytes induced by X-VIVO^TM 15 combined with TPO, SCF, G-CSF but without FBS were superior. And X-VIVO^TM15 medium was better than SCGM medium at effectiveness and cost. Using two-stage mode of hematopoietic stem cell expansion followed by granulocyte induction with X-VIVO^TM15 combining TPO, SCF and G-CSF, cell proliferation was nearly 132 times at day 21. Flow cytometry showed that the differentiation was lagged in 2-stage mode than in direct induction mode, CD15 expression was (69.60± 1.06) % vs (97.73±0.39) %; Wright-Giemsa staining demonstrated mature granulocytes; immunofluorescence showed the expression of lysosomal proteins CD18. A strong phagocytic function of mature granulocytes was demonstrated by phagotrophic efficiency of (51.43±0.05) %. And granulocyte had chemotaxis ability under the role of chemotactic factor IL-8. Conclusion Optimized culture media and cultivation mode are achieved for functional granulocytes induction in vitro.

Objective:To analyze the pathogenic bacteria and epidemiological characteristics in children with respiratory tract infection in Tianjin area.Methods:Retrospective case analysis was performed on 2 392 hospitalized children in the wards of respiratory diseases, intensive care unit and special care ward of Tianjin Children′s Hospital from June 2018 to May 2019. Thirteen pathogenic bacteria in deep sputum and bronchoalveolar lavage fluid samples were detected by loop-mediated isothermal amplification. The laboratory data and clinical characteristics of the infected children were analyzed, and the comparison between groups was performed by t test or χ 2 test. Results:Among 2 392 cases, 1 407 were males and 985 females. There was no significant difference in the detection rate between males and females (72.5% (1 020/1 407) vs.74.2% (731/985), χ 2=0.87, P=0.35). A total of 1 751 strains and 12 kinds of positive respiratory pathogens were detected, with a detection rate of 73.2%. Among them, 913 (38.2%) strains were Mycoplasma pneumoniae (MP), 514 (21.5%) were Streptococcus pneumoniae (Sp), 381 (15.9%) were Methicillin-resistant Staphylococcus aureus (MRSA) and 279 (11.7%) were Hemophilus influenzae (Hi). There was significant difference in the detection rate of pathogens among different age groups (χ2=83.67, P<0.01). The positive rate of alveolar lavage fluid group was higher than that of deep sputum fluid group [81.6% (614/752) vs. 69.3% (1 137/1 640), χ 2=39.89, P<0.01]. The length of hospital stay of children infected with different pathogens was significantly different (all P<0.01). There was significant difference in duration of fever among children infected with different pathogens (χ2=228.69,103.56, 3.96, 27.38,24.50,41.66, all P<0.05). There were 63 (7.7%) cases of atelectasis, 260 (31.9%) cases of pleurisy and 120 (14.7%) cases of pleural effusion in MP children. Children with Sma were most likely to involve the heart system (2/9), and children with Eco infection had a higher incidence of complications such as those of blood (3/19), urinary (2/19), digestive systems(4/19), systemic inflammatory response syndrome and sepsis (1/19). Conclusions:The main bacterial pathogens of respiratory tract infection in children in Tianjin were MP, Sp, MRSA and Hi. It is suggested that clinicians should not only pay attention to the respiratory symptoms of children, but also pay attention to the complications caused by bacterial pathogen infection, so as to prevent the deterioration of the disease and improve the prognosis.