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Objective To construct the genetic engineered cell line which can continuously secrete human tumor necrosis factor ( hTNFα) in Chinese Hamster Ovary cells( CHO cells) ,and observe the change of its protein secretion function.Methods Constructed plas-mid which carries hTNFαgene expression through vector GV141.Selected stable transfection cell lines by G418 and transfection with lipo-fectamine.Identified its gene expression with Real-Time PCR,and identified its protein secretion by ELISA.Results GV141-hTNFαexpres-sion vectors were constructed successfully which were proved by sequence alignment.Real-Time PCR proved that it contained hTNFαgene in hTNFα/CHO cell line.ELIAS identification results showed that the cell lines can continuously secrete hTNFαwithin a certain cell propaga-tion.Conclusion The hTNFα/CHO cell line can continuously secrete human tumor necrosis factor within a certain cell propagation.
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PurposeTo analyze the far infrared thermography characteristics before and after surgery in patients with acoustic neuroma.Materials and Methods Thirty-two patients with acoustic neuroma were included as observation group and 40 normal healthy individuals as control group. Un-cooled thermal imaging system (ATIR-M301B) was used with working temperature of 20-25℃. Craniofacial infrared images were collected to analyze temperature differences among different detection zones.Results Far infrared thermography revealed that there were no obvious temperature differences between both sides of supraorbital region, endocanthion region, frontal region and buccal region in the control group (P>0.05). The temperature differences in bilaterally symmetrical parts of supraorbital region, endocanthion region, frontal region, buccal region were significantly higher in observation group (t=1.557, 1.714, 1.483 and 1.569,P<0.05). The craniofacial temperatures of 32 patients changed after operation, and the differences reduced in supraorbital region, endocanthion region, frontal region, and buccal region (t=2.655, 2.462, 2.897 and 4.465,P<0.05).Conclusion Far-infrared thermography inspection can detect abnormal temperature changes.
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objective To observe the effects of co-culture of hTNFα-sercreting and human colon cancer cells LOVO on the proliferation of cancer cells. Methods The stable transfected hTNF-α/293 , mRNA of Hek-293 cell and protein expression were detected by RT-PCR and ELISA. The positive group was added hTNF-αfactor,and MTT assay was applied under the optical density 490 nm. Through human tumor cell proliferation inhibition experiment,the inhibitory effects on colon cancer cells ( LOVO) proliferation were observed. Results hTNF-α/293 cells and hTNF-α-positive group showed a significant lower A,which suggested that hTNF-α/293 cells and hTNF-α-positive group had significant inhibition on the proliferation of colon cancer cells. Conclusion The inhibition of hTNF-αsecreted by hTNF-α/293 cells on co-lon cancer cell proliferation shows significant dose-effect dependency,and hTNF-αexpresses a considerable inhibition on the colon cancer cell proliferation as positive drug.
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Objective This study aimed to understand the continuous physiological parameters during sleep under the guidance of Chinese medical theory and clinical practice and to establish the method to diagnose the physical and mental status by analyzing nocturnal sleep data.Methods More than 2000 subjects were recruited in the nocturnal sleep examination using the micro-movement sensitive mattress sleep monitoring system.Based on the analysis of the sleep indices and nocturnal physiological parameters combined with clinical data,a hypothesis was put forward that sleep cycle could reflect the circulation status of qi and blood,as well as some preliminary exploration on revealing the status of qi,blood,yin and yang.Results Sleep structure could reflect the status of qi and blood circulation in different meridians according to the traditional Chinese time unit.Sleep structure and sleep cycle could show corresponding changes if there is morbidity in some meridian,zang-viscera and fu-viscera.Heart rate,respiration rate and other physiological parameters could reflect the alternative predominance of yin and yang.Conclusion Interpreting the physiological parameters during sleep could be a supplementary diagnosis method for Chinese medicine syndrome differentiation.
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@#Objective To establish the model of glioma by hypodermic of C6 cells line into the nude mice and observe the growth characteristics. Methods The C6 gliocyte suspension was injected into the left subaxillary hypoderma of nude mice. The diameter and the volume of the tumor were measured and calculated, and the volume-time growth curve was plotted. The cells of tumor were observed under the HE and glial fibrillary acidic protein (GFAP) immono-histochemistry staining. Results The tumor appeared in inoculation area 7 d after injection, and presented nearly exponential development after about 20 d. The survival time of the nude mice is 22～48 d. The tumors were with sharp border, affluent blood vessel, and a great number of GFAP-positive cells. Conclusion The model of glioma can be well induced by hypodermic of the cultured C6 gliocyte into nude mouse.
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@#Objective To observe the effect of APA-BCC(alginate-polylysine-alginate microencapsulated bovine adrenal medullary chromaffin cells)microcapsules transplantation into the subarachnoid space of cancer pain patients on endogenous opioid peptides and catecholamine concentration in cerebrospinal fluid(CSF).Methods The different doses of APA-BCC microcapsules were transplanted into the spinal subarachnoid space of cancer patients with moderate or serious pain.The concentrations of Leu-enkephalin(L-EK),β-endorphin(β-EP),dynorphin A(DynA),noradrenaline(NA)and adrenaline(AD)in CSF were tested.Results L-EK concentration in CSF increased remarkably after transplantation,and the increase was most remarkable when transplantation doses were 1.0×107and 1.25×107;there were no remarkable changes of β-EP,DynA,NA and AD after transplantation.Conclusion The analgetic effects of APA-BCC microcapsules tranplantation may correlate with the increase of L-EK in CSF of cancer pain patients;the dose of 1.0×107 and 1.25×107cells may be the most effective dose.
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@#Objective To construct a cell line with long time secreting endostatin. Methods Human endostatin cDNA containing interleukin-2 (IL-2) secreting peptide was cloned into eukaryotic expression plasmid pSNA2.0 to construct recombinant plasmid pSNA2.0/Endostatin. The plasmid pSNA2.0/Endostatin was transfected into HEK293 cells by cationic liposome. The positive cell clones were selected by G418 and named hED/293. The expression of endostatin protein was analyzed by Western-blot. Results Endostatin could be determined in the supernatant of hED/293 cells. Conclusion The recombinant eukaryotic expression vector is correctly constructed. The human endostatin protein can be expressed and secreted.
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@#ObjectiveTo explore the methods of low temperature preservation for alginate-polylysine-alginate (APA) microcapsules.MethodsAPA microcapsules were prepared with static electricity, and underwent hypothermal treatment respectively through methods of program control, gradient by icebox and put in liquid nitrogen directly, finally preserved in liquid nitrogen. The form and permeability of APA microcapsules were checked after rewarming.ResultsThe rates of integrity, crenation and damage were (91.2±1.57)%, (3.1±0.81)% and (5.7± 2.62 )% in the program control group; (85.3±1.42)%, (5.2±0.74)% and (9.5± 3.81 )% in the gradient by icebox group; (14.5±1.57)%, (84.1±3.47)% and (1.4±2.62)% in the directly put in liquid nitrogen group. The membrane permeability of full APA microcapsules after frozen and reworming was not changed obviously.ConclusionThe program control method can preferably preserve APA microcapsules at low temperature and keep them having normal form and permeability.
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Objective:To study the immunoisolation effects of alginate-polylysine-alginate(APA)-microcapsules in vito.Methods:To observe changes of cellular immune and tectology of sheeps after hollow microcapsules, Bovine Chromaffin Cells(BCCs) and APA Microencapsulated BCCs(APA-BCCs) transplanted to the spinal subarachnoid space.Results:The blood lymphocyte numbers of each group didn't change; APA-microcapsule could prevent blood CD4 +T lymphocyte , CD4 +/CD8 + and CSF lymphocyte from increasing caused by BCCs transplantation; APA microencapsulation could reduce histological reactions in transplantation area and prolong the survival of the transplant.Conclusion:APA microcapsules possess the immunoisolation effects and can efficiently prevent immunoreactions.
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Objective To Observe the adjusting effect of bone morphogenetic protein 4 (BMP4) on cholinergic-expression in embryo rats' neurons. Method Neurons isolated from hippocampus and cerebral cortex of 15-20 days gestational age SD rat's brain were cultivated in a DMEM/F12 medium containing Ara-C and identified with morphological characters and microtubulin associated protein 2 (MAP2 ) immunocytochemical test. The medium was replaced with BMP4 96 hours after Ara-C adding, and cultivated another 10 days. The expression of choline acetyltransferase (ChAT) of the adjusted cells was observed by immunocytochemical staining and FITC-labeled cholinergic nerve cells Flow Cytometry. Results It has been showed that cultured neurons accorded with the typical morphological characters and the immunologic markers of neurons; the percentage of ChAT-positive cells in BMP4 group was prominently higher than that in control group(13.3?1.37% vs 6.44?0.81%, P
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Objective To establish a method of large-scale cryopreservation of porcine hepatocytes to meet the need of the biological artificial liver. Methods Porcine hepatocytes were isolated from Chinese miniature swines by collagenase disgestion. The cells were suspended in self-made nutrient solution supplemented with 10% DMSO. The hepatocytes (1?10 10~2?10 10) were stored in liquid nitrogen (-196℃) after being treated with two kinds of freezing methods. The cryopreserved hepatocytes were thawed in 39℃ water after one month and cultured. The morphology, viability and the function of thawed hepatocytes were observed. Results The cell viability of cryobreserved hepatocytes after both freezing methods were high. However, synthesis of urea nitrogen and glucose was higher in stepwise temperature lowering group compared with programmed temperature lowering group. Conclusion The results indicate that the cryopreservation method is feasible and simple, large amount porcine hepatocytes can be cryopreserved.
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AIM To invesigate the effect of subarachnoid transplantation of APA microcapsulized bovine chromaffin cells (BCCs) on mRNA expression for Nav1.8 in the dorsal root ganglia neurons(DRG) of rats with neuropathic pain by means of in situ hybridization. METHODS SD rats were randomly divided into four groups of five. Normal rats were used as control group (group C). Rats with right sciatic nerve been ligated were used as CCI group. Five to six hundred empty APA microcapsules(group APA) or 5?10 6 APA microcapsulized BCCs (group APA-BCCs) were grated into subarachnoid space of CCI rats 7 days after operation. Allodynia and hyperalgesia were measured by Von-Frey filaments and CO 2 laser 7 days after transplantation. DRG in lumbar four and five was taken out and 15 ?m freezing sections were made 7 days after tansplantation. Sections was used to detect mRNA expression for TTX-resistent Na + Nav1.8 by in situ hybridization with Dig-labeled RNA probe. RESULTS The mRNA hybridization signal for Nav1.8 in DRG of group CCI and group APA was lower than that of group C. The expression of mRNA for Nav1.8 in DRG was higher in group APA-BCCs than that in group CCI and group APA with abatement of allodynia and hyperalgesia. There was no difference in the mRNA hybridization signal for Nav1.8 in DRG between group APA-BCCs and group C. CONCLUSION mRNA expression for Nav1.8 in DRG of CCI ratswas down-regulated. APA microcapsulized BCCs grafting can reverse the down-regulation of mRNA expression for Nav1.8 in DRG of CCI rats. Restoration of mRNA expression for Nav1.8 in DRG contributes to the analgesic effect of subarachnoid transplantation of APA microcapsulized BCCs.
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Abstract To analyse the mechanism of TECA bio-artificial liver support system(BALSS)to treat acute liver failure (ALF) canines. The blood of partial liver resection-induced ALF canines or RPMI1640 solution were circulated in the inner space of the hollow fibers (blood loop), and porcine hepatocytes were cultured in outer space of the hollow fibers (cell loop). The concentrations of lidocaine, albumine and urine in the RPMI1640 solution were measured. The compositions of proteins in cell loop and blood loop were analysed by polyacrylamide gel electrophoresis (PAGE). The ultrastructure of porcine hepatocytes in BALSS was observed with transmission electronic microscopy. There were not any proteins in RPMI1640 solution before BALSS circulation. After 6 hours of the BALSS circulation, the concentration of lidocaine in RPMI1640 solution decreased significantly,and concentration of albumine and urine nitrogen increased gradually. The ultrastructure of the porcine hepatocyte shown damaged after BALSS 6h. The results suggested that TECA BALSS could replace temporarily the functions of failure liver by the effects of biologic transform and synthesis of porcine hepatocytes.
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Objective To assess the efficacy and safety of BioLiver Ⅰ bioartificial liver support system (BALSS) on the treatment of two types of acute liver failure (ALF) canine models. Methods The drug induced ALF canine model was established by multiple subcutaneous injections of acetaminophen. Another model was operated to resect 80 percent of the liver tissue. The primary hepatocytes were separated from the swine and cultured within the BALSS. The ALF models were treated by BioLiver ⅠBALSS for 6 hours. The changes of physiological, biochemical and histological parameters were observed before and after the treatment. Results The canines developed ALF 48 hours after injections of acetaminophen, the established rate of the model was 63 3%. While the other ALF canine model developed 24 hours after 80 percent liver resection and the established rate was 84 2%. Using our modified enzymatic digestion method, the yield of hepatocytes was (1 0～3 0)?10 10 per swine with high viability. BioLiver ⅠBALSS treatment resulted in beneficial effects on blood biochemical parameters. The pathological lesions of the liver were repaired. BALSS treatment was harmless to other organs. The ALF canines in drug group survived longer than in operation group. Conclusion This type of BALSS can provide safe and efficacious liver function support in the two types of ALF canine models, and it may be as an hepaful and important therapy to ALF