Objective:To evaluate the impact of altered collagen Ⅱ(CⅡ) peptide(S268-270) on T cell activation.Methods:Altered CⅡ263-272 with G268,P269 and K270 consecutively substitution with A or G were synthesized using solid phase various concentrations were added to HLADR1 transfected APC.Expression of FITC stainning was analyzed by fluorescence-activated cell sorting,and the binding of altered CⅡ peptide to HLA-DR1 molecule on cell membrane was evaluated.Irridiated HLA-DR1 expressing APC was incubated with CⅡ263-272 or hemagglutinin(HA)306-318 and altered CⅡ peptides at various concentrations for 2 hours before T cells(3.19) were added.Supernatant was harvested and then added to IL-2 dependent CTLL cell.The cell proliferation was examined by methotetrazolium(MTT) method.Results:The altered CⅡ peptide and wild type of CⅡ263-272 were able to competitively bind to HLA-DR1 molecule expressed on cell membrane of transfected APC.CⅡ or HA induced T cell activation was significantly inhibited by the altered peptide S268-270.Conclusion:Altered CⅡ263-272 with G268,P269 and K270 consecutively substitutions with A or G inhibited CⅡ263-272 and HA306-318 induced T cell activation through competitively binding to HLA-DR1,suggesting a new approach in inhibition of T cell activation in RA.

Objective To study the effects and mechanism of evodiamine on human colon cancer cell. Methods After human colon cancer SW620 cell were treated with different doses of evodiamine, the growth of anchorage independence of cancer cell was studied by colony formation in soft agar, and invasion ability was determined by Boyden chamber,and the level of mRNA and protein of midkine gene was detected by real time RT-PCR and Western blot assay, respectively. Results Ecodiamine could significantly inhibit both invasion ability and anchorage independence growth in dose-dependent manners. The level of mRNA and pro-tein of midkine of cancer cells treated with evodiamine reduced in time-and dose-dependent manners Conclusion Evodiamine could inhibit invasion of colon carcinoma cell through down-regulating of midkine expression.

Objective To explore the effects and mechanism of S100A4 gene silence on invasion of human thyroid cancer cell. Methods After thyroid cancer cell ARO was transfected by S100A4 small interfering RNA (siRNA), mRNA and protein level of S100A4 and matrix metalloproteinase 2 (MMP-2) were determined by real time RT-PCR and Western blot respectively. The anchorage-independent growth was examined by colony formation assay in soft agar, and invasion ability was evaluated by boyden chamber model. Results The level of mRNA and protein of S100A4 was significantly inhibited in ARO cancer cells transfected by S100A4 siRNA.Transfection with S100A4 siRNA could inhibit anchorage-independent growth and invasion ability of thyroid cancer cell ARO in a dose-dependent manner. mRNA and protein expression of MMP-2 were down-regulated by S100A4 siRNA. Conclusion S100A4 siRNA can inhibit the invasion of thyroid cancer cell through down-regulation of MMP-2.

Objective To study the burden of main earegivers of patients with stroke and its influ-encing factors, besides, the corresponding nursing countermeasures were disucssed. Methods 56 main caregivers of stroke families were selected to complete caregiver burden inventory (CBI). Results The general level of burden of main caregivers was in the moderate level with score value (1.55±0.35),factors influencing the burden of caregivers included ADL index, number of caregivers who involved in nursing and hospitalization times. Conclusions Nurses should educate the stroke patients about their rehabilitation systematically to decrease the severity of stroke sequela and readmission times, besides, they should strengthen their family support to reduce the burden of caregivers and increase their health status both physically and psychologically.

Objective To study the effects of cripto on migration, invasion, and liver metastasis of colorectal cancer cell. Methods After human colorectal cancer cell line SW480 was transfected by cripto small interfering RNA (siRNA), the mRNA and protein level were determined by Real-time RT-PCR and Western blot, respectively. The migration and invasion ability were evaluated by wound-healing assay and boyden chamber model, respectively. Thirty nude mice model of liver metastasis from colorectal cancer was established by splenectomy. Results The siRNA could down-regulate the level of mRNA and protein of cripto in a dose- and time-dependent manner. Suppression of cripto expression could inhibit migration and invasion ability of human colorectal cancer cell in vitro. The metastastic rate and tumor nodules were lower in transfection with cripto siRNA than in two control groups in vivo. Conclusions Cripto gene might play an important role in regulation of liver metastasis from colorectal carcinoma cell, and suppression of cripto gene by siRNA can inhibit liver metastasis of colorectal cancer.

Objective To study the impact of TDGF-1 gene silience by small interfering RNA(siRNA)on the invasion and migration of human breast cancer cell. Methods 3 siRNA fragments were designed according to the characteristic of TDGF-1 gene sequence and the most appropriate siRNA was selected by fluorescence real-time quantitative RT-PCR method. After the human breast cancer cell line MDA-MB-468 was transfected by the selected TDGF-1 siRNA, mRNA and protein of TDGF-1 were determined by real time quantitative RT-PCR and western blot respectively. The migration and invasion ability of the cancer cell were evaluated by wound-healing assay and Boyden chamber model respectively. Results siRNA could down-regulate the level of mRNA and protein of TDGF-1 in a dose-and time-dependent manner. In vitro experiment showed that TDGF-1 siRNA transfection can effectively inhibit the clonal growth, invasion and migration of breast cancer cell in a dose-dependent manner. Conclusions TDGF-1 gene may play an important role in the migration and invasion of human breast cancer cells. siRNA transfection can inhibit the invasion of human breast cancer cells.

Objective To investigate the effects of S100A6 gene on invasion of human pancreatic cancer cell and possible mechanism. Methods Human pancreatic cancer BxPC3 cell line was transfected with small interfering RNA (siRNA) targeting S1006 gene, the mRNA and protein levels of S100A6 were determined by real time RT-PCR and Western blotting respectively. The invasion ability was evaluated by Transwell chamber. The matrix metalloproteinase-2 (MMP-9) activity of cancer cells was examined by gelatin zymography. Results The levels of mRNA and protein of S100A6 were greatly reduced in a dose and time dependent manner, the number of penetrating cells was greatly reduced in a dose dependent manner. The expression of S100A6 mRNA in 12.5 nmol/L of S100A6 siRNA transfected group decreased from ( 100 ±0.3)％ in control group to (15.3 ±0.2)％ ; while the expression of S100A6 protein decreased from (83.2 ±0. 18 ) ％ to ( 13.5 ± 0. 12) ％ ; the number of penetrating cells decreased from 44.5 ± 2.2 to 7.6 + 1.5 ( P ＜0. 01 ). The MMP-9 activity of siRNA group reduced significantly. Conclusions S100A6 siRNA can inhibit the invasion of pancreatic cancer cells through down-regulation of MMP-9.

Objective To study the effects VEGF small interfering RNA (siRNA) on chemosinsitivity of human BxPC3 cell and its mechanism. Methods BxPC3 cells were divided into single BxPC3 cell group,lipofection group, scrambled siRNA transfection (200 nmol/L) group, VEGF siRNA transfection group.VEGF siRNA (5, 10, 20, 100,200 nmol/L) was used to transfect BxPC3 cells. Expressions of VEGF mRNA and protein were determined by real-time PGR and ELISA assay, respectively. MTT was performed to examine the inhibitory effects of gemcitabine on BxPC3 cells of each group. The phosphorylated-Akt protein was evaluated by Western blotting. Results After VEGF siRNA transfection, the expression of VEGF mRNA and protein in BxPC3 cells was down-regulated in a dose-and time-dependent manner, but there was no effect on BxPC3 cells proliferation. After 0. 2 μmol/L of gemcitabine treatment for 48 h, the inhibitory rates were ( 16.9 ±0.3)%, (17.3 ±0.3)%, (28.8 ±0.4)%, (52.2 ±0.3)%, (75.4 ±0.4)% in BxPC3 cell group,lipofection group, 5,10,20 nmol/L VEGF siRNA transfection group, and the inhibitory effects were correlated with siRNA concentration ( r = 0. 928 ). The phosphorylated-Akt protein was reduced significantly in siRNA transfected cells. Conclusions VEGF gene plays an important role in BxPC3 cells resistence to chemotherapy through inhibiting Akt phosphorylation.

Objective To study effects of polo-like kinase-1(PLK1)small interfering RNA(siRNA)on proliferation and apoptosis of undifferentiated human thyroid cancer cells.Methods 5 PLK1 siRNA(S1,S2,S3,S4 and S5)were constructed and used to transfect human thyroid cancer cell line ARO.RT-PCR was employed to pick out the most effective siRNA,which was then used to transfect ARO cell.RT-PCR and western blot were used to detect PLK1 expression in thyroid cancer cells,which were divided into different groups.MTT assay was performed to examine the effects of PLK1 siRNA on thyroid cancer cells in all groups.Apoptosis of thyroid cancer cells was observed by caspase-3 activity and TUNEL.Results All the 5 siRNA down-regulated PLK1 mRNA expression.among which S4 showed the best effect.S4 transfection could obviously inhibit proliferation of thyroid cancer cells in dose and time dependent manner.Compared with control groups,caspase-3 activity of cancer cells in s4 transfeeted group increased significantly.The effect of S4 transfection was dose and time dependent.TUNEL results showed apoptosis of cancer cells transfected by S4 siRNA was obvious and apoptosis of cells was dose-dependent.Conclusions PLK1 may play an important role in proliferation of undifferentiated thyroid carcinoma.PLK1 siRNA transfection can inhibit proliferation of throid cancer cell through apoptosis induction.

Objective To study the effects of midkine(MK)gene small interfering RNA(siRNA)on adhesion and invasion of human breast cancer cells.Methods Real time PCR was used to evaluate MK mRNA expression in 7 human breast cancer cell lines Bcap-37,LCCI,MCF-7,MDA-MB-231,MDA-MB-435,MDA-MB-468,and ZR75-1.The cell line in which MK expression was the highest was transfected with different doses of MK siRNA.The expression of MK mRNA and protein was determined by real-time quantitative PCR and immunoflurescence staining.The cell adhesion was evaluated by MTT assay and invasion was examined by Boyden chamber method.Results Cell line MCF-7 expressed the highestlevel of MK mRNA in the 7 tested breast cancer cell lines.After being transfected with MK siRNA,MK mRNA and protein level of MCF-7 decreased in timeand dose-dependent manners.The adhesive and invasive ability of MCF-7 cell transfected with MK siRNA decreased in a dose dependent manner(P<0.01,P<0.01).Conclusions MK gene might play an important role in adhesion and invasion of human breast cancer cells.siRNA transfection could effectively inhibit adhesion,migration,and invasion of human breast cancer cell.