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.

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.

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.

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.

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.

OBJECTIVETo quantitatively analyze the temporal and spatial pattern of RhoA expression in injured spinal cord of adult mice.

METHODSA spinal cord transection model was established in adult mice. At 1, 3, 7, 14, 28, 56 and 112 days after the surgery, the spinal cords were dissected and cryosectioned for RhoA/NF200, RhoA/GFAP, RhoA/CNPase or RhoA/IBA1 double fluorescent immunohistochemistry to visualize RhoA expressions in the neurons, astrocytes, oligodendrocytes and microglia. The percentages as well as the immunostaining intensities of RhoA-positive cells in the parenchymal cells were quantitatively analyzed.

RESULTSRhoA was weakly expressed in a few neurons and oligodendrocytes in normal spinal cord. After spinal cord injury, the percentage of RhoA-positive cells and RhoA expression intensity in the spinal cord increased and peaked at 7 days post injury (dpi) in neurons, oligodendrocytes and astrocytes, followed by a gradual decrease till reaching a low level at 112 dpi. In the microglia, both the RhoA-positive cells and RhoA expression intensity reached the maximum at 14 dpi and maintained a high level till 112 dpi.

CONCLUSIONTraumatic spinal cord injury can upregulate RhoA expression in the neurons as well as all the glial cells in the spinal cord. RhoA expression patterns vary with post-injury time, location and among different parenchymal cells in the injured spinal cord.

Animals ; Astrocytes ; metabolism ; Female ; Mice ; Mice, Inbred Strains ; Microglia ; metabolism ; Neuroglia ; metabolism ; Neurons ; metabolism ; Spinal Cord ; metabolism ; Spinal Cord Injuries ; metabolism ; rho GTP-Binding Proteins ; metabolism