[Effects of Hedyotis diffusa Extract on Epidermal Growth Factor Induced Proliferation, Apoptosis, and TNF-α Induced Inflammatory Factors of HaCaT Cells].

Objective To observe the effects of Hedyotis diffusa extract (HDE) on the prolifera- tion, apoptosis, and inflammatory factors of HaCaT cells, and to explore its possible molecular mecha- nisms. Methods HaCaT cells were divided into 3 groups, the vehicle group, the control group, and 3 dose HDE groups. No epidermal growth factor (EGF) or HDE was added in the vehicle group. EGF was added with no HDE treatment in the control group. HDE (25, 50, 100 mg/mL) and EGF were added in the 3 HDE groups to co-culture HaCaT cells. The effects of HDE on EGF-induced proliferation of HaCaT cells were de- tected using CCK-8 assay. The effects of HDE on the growth cycle and apoptosis rate of HaCaT cells were measured using flow cytometry. Moreover, protein expression levels of Ki67, Bcl-xL, clAP1 , procaspase- 3, and cleaved caspase-3 were determined using Western blot. In addition, levels of IL-6, IL-8, and IL-10 in the supernatant were detected using ELISA. The level of phosphoration of NF-κB p65 (p-p65) was meas- ured using Western blot. Results Compared with the vehicle group, the absorbance of HaCaT cells and the expression level of Ki67 increased (P <0. 05, P <0. 01) ; levels of p-p65, IL-6, and IL-8 were elevated (P <0. 05, P <0. 01); IL-10 level was lowered (P <0.01) in the control group. Compared with the control group, the absorbance of HaCaT cells and the expression level of Ki67 decreased (P <0.05, P <0.01) ; levels of p-p65, IL-6, and IL-8 were reduced (P <0. 05, P <0. 01); IL-10 level was elevated (P <0. 05, P < 0.01) in the 3 dose HDE groups. Meanwhile, the apoptosis rate of HaCaT cells increased more in the 3 dose HDE groups than in the control group (P <0. 05, P <0. 01). The percentage of HaCaT cells at G1 phase was 58. 51 %, 73.12%, and 79. 95% in 25, 50, and 100 mg/mL HDE groups, respectively, showing statisti- cal difference when compared with that in the control group (52. 85%; P <0. 05, P <0. 01). The percentage and apoptosis rate of HaCaT cells at G1 phase were elevated more in 50 and 100 mg/mL HDE groups than in 25 mg/mL HDE group (P <0. 01). Besides, the percentage and apoptosis rate of HaCaT cells at G1 phase were elevated more in 100 mg/mL HDE group than in 50 mg/mL HDE group (P <0. 05). Compared with the control group, protein expression levels of Bcl-xL and cIAP1 were reduced in 100 mg/mL HDE group (P < 0. 01). There was no statistical difference in caspase-3 total amount (P >0. 05), but cleaved caspase-3 ex- pression increased with statistical difference (P <0. 01). Conclusion HDE inhibited the proliferation of HaCaT cells possibly by arresting HaCaT cell growth at G1 phase, promoted the apoptosis of HaCaT cells by stressing protein expressions of Bcl-xL and cIAPI , and elevating protein expressions of cleaved caspase-3, and suppressed inflammatory response of HaCaT cells via regulating NF-κB expression.

[Establishment of the tax gene expression cell lines and their biological activity].

This study was aimed to establish the cell lines of human T-cell leukemia virus type-1 (HTLV-1) tax gene expression and their biological activity. The eukaryotic vector pCMV-tax and pCMV-neo-Bam were transfected into Jurkat E6-1 by using liposome, following screening with G418, the tax gene expression cell line-TaxP and the negative cell lines-TaxN were selected and enriched. Then, the mRNA expressions of LAT, SLP70, ZAP70 and NF-kappaB (p65) were measured by using RT-PCR; the NF-kappaB bioactivity was tested after transfecting the pNF-kappaB-Luc plasmids into TaxP and TaxN cells; meanwhile CD25, CD69 expressions on surface of the two cell lines were tested by flow cytometry. The results showed that TaxP and TaxN cell lines were established and the tax gene expression was detected; as compared with TaxN, the mRNA expressions of LAT, SLP70 and ZAP70 in TaxP were enhanced (p<0.05), while the NF-kappaB bioactivity in TaxP cells was stronger than that in TaxN cells (p<0.01). The CD25, CD69 on TaxP cells surface was highly expressed. It is concluded that TaxP and TaxN cell lines are established, TAX protein could promote the activation of T cells and activate the NF-kappaB pathway.

[Expression of human growth hormone gene in mice and its effect on concentration of murine serum cytokines].

AIM: To explore the effects of high level human growth hormone(GH) on immune function. METHODS: The eukarytic expression plasmid containing hGH gene was constructed and then was injected into musculi quadriceps femoris of mice. 5th, 15th and 25th day after injection, the murine sera were collected and the levels of hGH, IL-2, IFN-gamma and TNF-alpha were detected by ELISA. RESULTS: hGH cDNA was correctly cloned into pcDNA3. COS-1 cells transfected with the recombinant plasmid secreted high level of active hGH. The concentration of hGH in the sera of mice receiving the plasmid was obviously higher than that of control group. However, the concentrations of IL-2, IFN-gamma and TNF-alpha had no significant variation in comparison with those of control group. CONCLUSION: High level GH has no marked effect on secretion of IL-2, IFN-gamma and TNF-alpha.