Serum is an indispensable factor in the maintenance of the biological characteristics of sweat gland cells.

The tolerance of sweat gland cells for in vitro amplification and subcultivation is low as they are somatic cells. The present study aimed to formulate an optimal medium for the culture of human eccrine sweat gland cells (HESGCs) and to establish a method for induction of HESGCs proliferation, whilst maintaining the characteristics of sweat gland cells. HESGCs cultured in sweat gland (SG):keratinocyte growth medium­2 (KGM­2) (1:1) medium had a higher proliferation rate and a stable morphology compared with cells cultured in SG and KGM­2 medium only. Reverse transcription­quantitative polymerase chain reaction indicated that cells cultured in the SG:KGM­2 (1:1) medium exhibited higher expression levels of α­smooth muscle actin, keratin (K)77, carcinoembryonic antigen, K8, K18, ectodysplasin A receptor, c­Myc, Kruppel­like factor 4 and octamer­binding transcription factor 4 compared with cells cultured in SG only or KGM­2 only medium. Three­dimensional culture analysis revealed that HESGCs cultured in SG:KGM­2 1:1 medium differentiated into sweat gland­like structures, whereas cells cultured in KGM­2 only medium underwent cornification. The present study also determined that the maintenance of the biological characteristics of HESGCs occurred due to the presence of fetal bovine serum (FBS). Cells cultured in medium without FBS differentiated into keratinocytes. Therefore, the SG:KGM­2 (1:1) medium may be a suitable culture medium for HESGCs. In conclusion, this mixed medium is a valuable compound and should be considered to be a potential supplemental medium for HESGCs.

Clinical study on the treatment of knee osteoarthritis of Shen-Sui insufficiency syndrome type by electroacupuncture.

OBJECTIVE: To study the clinical effificacy of electroacupuncture (EA) on treating knee osteoarthritis (KOA) of Shen ()-Sui () insuffificiency (SSI) syndrome type. METHODS: A total of 245 patients (279 knees) of KOA-SSI were randomly assigned to two groups by lottery: 141 knees in the treatment group and 138 knees in the control group. The treatment group was managed with EA at the dominant points of Neixiyan (Ex-LE4) and Waixiyan (Ex-LE5) as well as the conjugate points of Xuanzhong (GB39) and Taixi (KI3) for 30 min, once a day, with 15 days as one course; 2 courses were applied with a 5-day interval in between. The control group was treated with intra-articular injection of 2 mL hyaluronic acid into the affected joint every 7 days for 5 times in total. The clinical effects on the patients in different stages were observed, and their symptom scores of knee and contents of cytokines, including interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), prostaglandin E(2alpha) (PGE(2alpha)) and matrix metalloproteinases-3 (MMP-3), in the knee joint fluid were measured before and after treatment. RESULTS: The study was completed in 235 patients (263 knees); four patients (7 knees) in the treatment group and six patients (9 knees) in the control group dropped out. Comparison of therapeutic effects (excellent and effective rates) between the two groups showed insignificant differences (P>0.05). Symptom scores of knee and contents of cytokines in the knee flfluid after treatment were lowered signifificantly in the patients of stage I-III in both groups (P<0.05 or P<0.01). However, the lowering of the total symptom score of knee in the patients of stage III in the treatment group was more signifificant (P<0.05). CONCLUSIONS: EA could effectively alleviate the clinical symptoms in KOA patients of stage III, showing an effect superior to that of hyaluronic acid. EA also shows action in suppressing the secretion of IL-1, IL-6, TNF-alpha, PGE(2alpha) and MMP-3 in the knee flfluid.

[Effect of shock lymph on the expression of inflammatory mediators in pulmonary micro-vascular endothelial cells of rats].

OBJECTIVE: To observe the effect of lymph collected during shock on free radical and expressions of nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) mRNA of pulmonary micro-vascular endothelial cells (PMVECs) of rats in order to explore the mechanisms of damaging effect of lymph collected during shock to the PMVECs. METHODS: PMVECs were isolated and cultured, and used at passage 3. The model of serious hemorrhagic shock was reproduced by maintaining the arterial blood pressure of rats at 40 mm Hg (1 mm Hg=0.133 kPa) for 90 minutes by exsanguination under aseptic condition. Mesentery lymph and portal vein blood were obtained from both shock rats and normal rats. PMVECs were respectively incubated in them for 6 hours, and at the same time, fetal bovine serum (FBS) and serum-free DMEM were used as culture media for comparison. The expressions of inducible nitric oxide synthase (iNOS), TNF-alpha and IL-6 mRNA were detected by the method of reverse transcription-polymerase chain reaction (RT-PCR), and the content of malondialdehyde (MDA), NO, TNF-alpha and IL-6 in culture supernatants were determined. RESULTS: After the PMVECs was treated by shock lymph at a final concentration of 4% for 6 hours, the expressions of iNOS, TNF-alpha and IL-6 mRNA in PMVECs and the contents of MDA, NO, TNF-alpha and IL-6 in culture supernatant fluids in shock lymph group were significantly increased compared with those of FBS group, normal lymph group, shock plasma group, normal plasma group and DMEM group. At the same time, the expressions of iNOS, TNF-alpha and IL-6 mRNA in PMVECs and the contents of NO in culture supernatant fluid of shock plasma group were significantly increased compared with those of FBS group, normal lymph group, normal plasma group and DMEM group (P<0.05 or P<0.01). CONCLUSION: The results demonstrate that the shock lymph in final concentration of 4% could enhance the expressions of iNOS, TNF-alpha and IL-6 of PMVECs, reduce the free radical, and as a result, induce damage to PMVECs.

[Effect of ligation of mesenteric lymph duct on inflammation response of liver in hemorrhagic shock in rats].

OBJECTIVE: To observe the effect of ligation of mesenteric lymph duct on changes in free radicals and pro-inflammatory mediators in the liver of rats with serious hemorrhagic shock at different periods, and explore the effect of blockage of intestinal lymphatic pathway on inflammation response of liver. METHODS: Seventy-eight male Wistar rats were randomly divided into the sham group (n=6), shock group (n=42), and ligation group (n=30). The model of serious hemorrhagic shock was reproduced in shock group and ligation group. Mesenteric lymph was blocked by ligating mesenteric lymph duct in ligation group after resuscitation. Six rats were sacrificed at 0, 1, 3, 6, 12, and 24 hours, and the livers were harvested and homogenized for the determination of malondialdehyde (MDA), nitric oxide (NO), nitric oxide synthase (NOS), superoxide dismutase (SOD), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and myeloperoxidase (MPO) activity. The expression of inducible nitric oxide synthase (iNOS) mRNA in liver was detected by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The contents of TNF-alpha, IL-6, NO, NOS, MDA, MPO and iNOS mRNA in liver homogenate of shock group were increased after transfusion and resuscitation, and their levels were higher at 6 and 12 hours. The values were significantly higher than those of the sham group, while the activity of SOD was significantly lower than that of sham group (P<0.05 or P<0.01). The contents of TNF-alpha, IL-6, NO, NOS, MDA, MPO and iNOS mRNA in liver homogenate were lower significantly after transfusion and 3, 6, 12 and 24 hours after resuscitation than those of shock group at each time points, and the SOD activity was higher (P<0.05 or P<0.01). CONCLUSION: The results demonstrate that the ligation of mesenteric lymph duct could reduce the polymorphonuclear leucocyte (PMN) detain, and its mechanism might relate to reduction of neutrophil aggregation, thus decreases the release of TNF-alpha and IL-6, reduces the NO and expression of iNOS mRNA, reduces the release of free radicals and consumption of SOD, as a result, it reduces the inflammation response of liver in serious hemorrhagic shock rats.

[Effects of shock lymph inducing the mesentery micro-lymphatic endothelial cells apoptosis of rats].

The model of serious hemorrhagic shock was established in the condition of asepsis and mesentery lymph was taken out. As control, normal mesentery lymph fluid, normal portal vein blood and shock portal vein blood of rats were taken out. The primary mesentery micro-lymphatic endothelial cells (MMLECs) of passages 3 were treated by different treatment factors, respectively. The cells proliferation by shock lymph and normal lymph of different final concentrations was measured using MTT method. The cell cycle arrest was analyzed by flow cytometry, and the electrophoresis analyze on DNA of cell nucleus was observed. The expressions of relative genes of apoptosis such as fas, fasL, bcl-2 and bax of MMLECs were detected by RT-PCR. The results showed that the MMLECs proliferation was decreased when the concentration of shock lymph increased to some extent and showed statisticly significant difference compared with normal lymph group. The cells cocultured with shocking lymph fluid at 4% final concentration showed that apoptosis rate of MMLECs was (35.4 +/- 1.6)%, and G0-G1 cell population was higher and the proportion of S+G2-M cell population was lower than that of other groups, and the DNA ladder was observed in electrophoresis of cell nucleus DNA at same time. And the expressions level of fas, fasL and bax mRNA were higher and bcl-2 mRNA was lower in shock lymph group than that of control group. The results demonstrated that shock lymph could reduce the cells proliferation, interfere with cell cycle, and accelerate high expression of apoptosis accelerative genes. As a result, shock lymph could induce the MMLECs apoptosis.

[Effect of tetromethylpyrazine and aminoguanidine on expression of vascular endothelial growth factor in kidney of diabetic rats].

OBJECTIVE: To investigate the therapeutic mechanism of tetromethylpyrazine and aminoguanidine on diabetic nephropathy. METHODS: Diabetic rats were induced by streptozotocin. They were divided into 5 groups: normal control group (group C), untreated diabetic group (group DM), tetromethylpyrazine treated group (group TMP), aminoguanidine treated group (group AG) and tetromethylpyrazine and aminoguanidine treated group (group TMP+AG). The expression of vascular endothelial growth factor (VEGF) in renal cortex of the rats in each group was observed by immunohistochemical staining after 12 weeks. RESULTS: The expression of VEGF in renal cortex of the rats in group TMP+AG and group C was alike. The expression of VEGF in renal cortex of group TMP and group AG decreased significantly as compared with that of group C, but was still above normal level. CONCLUSION: The therapeutic mechanism of tetromethylpyrazine and aminoguanidine on diabetic nephropathy may be inhibiting the over-expression of VEGF in kidney of diabetic rats.