[Expression of integrin-linked kinase in fibroblasts of scar induced by cobalt chloride and its effect on cell proliferation].

OBJECTIVE: To explore the expression of integrin-linked kinase (ILK) in fibroblasts (Fbs) of scar induced by cobalt chloride (CoCl2) and its effect on cell proliferation. METHODS: The human hypertrophic scar Fbs of seven patients were isolated and cultured in vitro. Cells from the 5th to the 6th passages were used in the experiment. Six bottles of Fbs were obtained from each of the seven patients, and they were respectively cultured with DMEM nutrient solution containing CoCl2 in the concentration of 0, 50, 100, 150, 200, and 250 µmol/L for 24 h. The expression of ILK mRNA was determined with real-time fluorescence quantitative PCR. Fbs were stimulated by CoCl2 in the most suitable concentration (100 µmol/L) and the protein expression of ILK was determined 0, 1, 2, 4, 12, and 24 h after the stimulation. Then the Fbs were divided into control group (cultured with nutrient solution), negative control group (transfected with con-siRNA), and ILK siRNA group (transfected with ILK siRNA). They were cultured with nutrient solution containing CoCl2 in different concentrations 24 h after transfection, with 4 wells for each concentration in each group. The cell proliferation was detected by XTT assay. Data were processed with one-way analysis of variance (ANOVA) and ANOVA for repeated measurement, and LSD method was used in multiple comparisons. RESULTS: The expression level of ILK mRNA was highest in Fbs cultured with 100 µmol/L CoCl2 for 24 h, with significant difference compared with those of Fbs cultured with other concentrations of CoCl2 (F = 50.958, P < 0.001). The expression of ILK protein in Fbs cultured with 100 µmol/L CoCl2 for 1 h (0.243 ± 0.009) was lower than that cultured for 0 h (0.387 ± 0.017), and it started to increase from 2 h (0.361 ± 0.010), and exaggerated at 4 h (0.584 ± 0.028), 12 h (0.730 ± 0.029), and 24 h (0.785 ± 0.031). The expression levels of ILK protein at 1, 4, 12, 24 h were statistically different from that at 0 h (P values all below 0.05). XTT showed that cell proliferation level was highest in control group when cultured with 100 µmol/L CoCl2 (F = 488.026, P < 0.001), which decreased from 150 µmol/L. The cell proliferation level in control group cultured with 250 µmol/L CoCl2 was significantly lower than that with 0 µmol/L (P values all below 0.05). There was no significant change in cell proliferation in ILK siRNA group among different concentrations of CoCl2 (F = 2.542, P = 0.056). The cell proliferation level in ILK siRNA group was significantly lower than that in control group and negative control group (F = 2519.542, P < 0.001). CONCLUSIONS: ILK may be a key protein in response of hypoxia in Fbs. The mild hypoxia can stimulate the expression of ILK and promote the proliferation of Fbs, while severe hypoxia can reduce the expression of ILK and inhibit cell proliferation.

[The effects of intergrin-linked kinase on angiogenesis in hypertrophic scar].

OBJECTIVE: To investigate the effects and regulatory mechanism of ILK on angiogenesis in hypertrophic scar. METHODS: The human scar microvascular endothelial cells (HSMECs) were isolated from 6 patients' hypertrophic scar in vitro. The HSMECs with good condition in 2nd to 4th generation were selected as experimental objectives. (1) HSMECs were divided into the blank control group (treated with routine culture), negative control group (treated with only Lipofectamine 2000), LY294002 group (incubated with 50 nmol/L LY294002), ILK siRNA group (incubated with 20 nmol/L ILK siRNA). RT-PCR and Western Blot were used to detect the expression of ILK mRNA and its protein after transfecion for 48 h. (2) The digested HSMECs of four groups were resuspended with DMEM without serum and then seeded onto the upper compartment of transwell insert which contained complete medium in its lower compartment. The cell migration experiment was stopped in 10 h and then the migrated cells were counted to analyze the effects of different interventions on the migration ability of HSMECs. (3) The thawed ECMatrix was put into each well of pre-colled 48-well tissue culture plate, and then the plate was put into the incubator at 37 degrees C to make it to become gel. The HSMECs of four groups were seeded onto the surface of the ECMatrix gel and were put into incubator. Eight random view-fields per well should be valued by the sheet of pattern recognition about angiogenesis after 8 hours to evaluate the ability of angiogenesis in vitro between four groups. RESULTS: (1) The expression of ILK mRNA (ILK mRNA = 0.829 +/- 0.109, t = 13.151, P = 0.006) and protein (ILK protein = 0.096 +/- 0.049, t = 36.656, P = 0.000) were both inhibited obviously in ILK siRNA group compared with the blank control group (ILK mRNA = 0.829 +/- 0.109, ILK protein = 1). And, the expression of ILK in LY294002 group was slightly lower than that of black control group, but there was no statistical difference. (2) The number of migrated cells in ILK siRNA group (88.111 +/- 3.079) and LY294002 group (138. 667 +/- 2.404) were respectively lower than that in blank control group (322.333 +/- 3.712, P < 0. 05) in 10th hour. (3) Compared to blank control group (4.333 +/- 0.191), the ability of angiogenesis in vitro decreased significantly ILK siRNA group (2.625 +/- 0.125) and LY294002 group (3.125 +/- 0.250), in which, the vascular network structures were not formed perfectly in 8th hour (P < 0.05). CONCLUSIONS: The ability of HSMECs' migration and angiogenesis in vitro are inhibited significantly when the expression of ILK is down-regulated. It reveals that ILK may play an role in the regulation of scar angiogenesis.

[Expression of integrin-linked kinase in human hypertrophic scar and its relationship with angiogenesis].

OBJECTIVE: To explore the expression of integrin-linked kinase (ILK) in scar in different growth stages, as well as its relationship with angiogenesis. METHODS: (1) Fifteen burn patients with scar formation time shorter than 6 months, ranging from 6 to 12 months, and longer than 12 months were hospitalized from December 2009 to December 2010. They were divided into A, B, and C groups according to the scar formation time, with 5 patients in each group. Scar specimens were harvested for observation of ILK expression with immunohistochemistry method, and ILK mRNA expression with real time fluorescence quantitative RT-PCR. (2) Microvascular endothelial cells (MEC) were isolated from scar tissue in A group and cultured in vitro, and then they were purified by immunomagnetic beads and identified with coagulation factor VIII marked by immunofluorescence (fibroblasts from human normal skin were used as control). The cultured cells in logarithmic growth phase were divided into control group (cultured with M131 medium containing microvascular growth supplement), transfection 1 group (transfected with empty plasmid), and transfection 2 group (transfected with ILK cDNA plasmid) according to the random number table. After 24 hours, the expressions of ILK mRNA, Flt-1 mRNA, and KDR mRNA were determined with real time fluorescence quantitative RT-PCR. Data were processed with one-way analysis of variance. RESULTS: Immunohistochemical observation showed that ILK in A group mainly expressed in the basal layer cells of epidermis, cytoplasm of fibroblasts, and MEC in scar, while ILK in B group only distributed in the basal layer cells of epidermis, but ILK expression in C group was not obvious. The expression of ILK mRNA in A group (0.34 ± 0.16) was significantly higher than those in B and C groups (0.17 ± 0.06, 0.07 ± 0.13, F = 37.007, P = 0.000). MEC grew up showing cobble stone formation after purification. The expression of coagulation factor VIII was positive in cytoplasm of purified MEC, while that was negative in fibroblast of human normal skin. The expressions of ILK mRNA (57.807 ± 5.556), KDR mRNA (0.836 ± 0.014), and Flt-1 mRNA (0.162 ± 0.005) in transfection 2 group were higher than those in control and transfection 1 groups (0.018 ± 0.003, 0.028 ± 0.020, 0.023 ± 0.004 and 0.042 ± 0.005, 0.039 ± 0.007, 0.046 ± 0.003; F(ILK) = 87.110, F(KDR) = 11.241, F(Flt) = 18.199, with P values all below 0.01). CONCLUSIONS: ILK mainly expressed in scar tissue with formation time shorter than 6 months, and it may affect vascularization of scar by regulating gene expressions of KDR and Flt-1 in MEC, which plays an important role in early scar formation.

Experimental study of the effect of human cytomegalovirus infection on cell cycle progression and the expression of cyclins / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To observe the effect of human cytomegalovirus infection on the host cellular DNA synthesis and expression of cyclones.</p><p><b>METHODS</b>HCMV infected cell was established in vitro by incubating passage cultured HEL and HCMV AD169 strain with different titres. The cells were synchronized in the G0/G1 stage by contact inhibition and infected with strain AD169 of HCMV at an MOI of 5 PFU per cell. We harvested infected cell at different time 0 h, 3 h, 6 h, 24 h, 72 h and 96 h post infection. Then the cell cycle progress was measured. Meanwhile, the DNA content and expression of proteins of cycline E, cycline A and cycline D1 were determined with FCM and Western Blot respectively.</p><p><b>RESULTS</b>We found that the amount of S stage cell infected by HCMV had increased dramatically, and that of G2/M stage cell reduced during 24 h-96 h PI, and no G2/M stage cell was detected within 96 h PI. The content of 2N DNA maintained unchangeable for 24 h after infection and the content of total DNA in infected cells began to increase within 48 h PI, and the substantial cell with 2N DNA were observed 72 h after infection. However, DNA content was not altered in control group of normal HEL and HCMV PAA group. CyclinE protein was induced 12 h PI and peak induction occurred 24 h PI in contact-inhibited cells. CyclinA protein expression was not induced in HCMV infected density-arrested cells. The abundance of cyclinD1 decreased 24 h PI.</p><p><b>CONCLUSION</b>The expression of cyclinE and activity of cyclinE/Cdk2 kinase are increased obviously in G0/G1 stage cells infected with HCMV, which may induce the cell cycle to overpass G1/S restriction point and make the cell cycle arrested in later G1 stage. HCMV can not activate cellular DNA synthesis, and increase of total DNA content in infected cells result from the viral DNA replication.</p>

Effects of Jinye Baidu Granule on fetal growth and development with maternal active human cytomegalovirus infection / 中国结合医学杂志

<p><b>OBJECTIVE</b>To evaluate the effects of Jinye Baidu Granule ( JYBDG), a traditional Chinese medicine compound prescription, on fetal growth and development with maternal active human cytomegalovirus infection.</p><p><b>METHODS</b>A prospective, randomized and controlled trial was adopted during January 1996 to June 2002. From the pregnant women with an abnormal pregnant history, 240 cases were screened to be infected by human cytomegalovirus (HCMV) by enzyme-linked immunoabsorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR). They were assigned according to the random number table to two groups. The 122 cases in the treatment group were administrated with JYBDG, one package each time, three times a day for two continuous weeks, while the other 118 in the control group did not receive any treatment. The negative conversion rate of both HCMV-IgM and HCMV late mRNA, the positive rate of HCMV-DNA in placenta and the intrauterine transmission rate between the two groups were compared, and fetal growth and development in partial fetuses were also observed.</p><p><b>RESULTS</b>The negative conversion rate of both HCMV-lgM and HCMV late mRNA, the positive rate of HCMV-DNA in placenta and the intrauterine transmission rate in the treatment group were 77. 05% (94/122), 48. 98% (48/98) and 21.74% (10/46) respectively, while those in the control group were 38. 14% (45/118), 67.50% (54/80) and 52.63% (20/38) respectively, all showing significant difference between the two groups (P<0. 01). Totally 35 normal infants and 11 abnormal infants were born in the treatment group, and the number in the control group was 20 and 18 respectively, and comparison between the two groups showed significant difference (P<0.01). Six months of child birth, the scores of both mental development index (MDI) and psychomotor development index (PDI) of infants were higher in the treatment group (20 cases) than those in the control group (20 cases), but there was no significant difference between the two groups (P>0.05).</p><p><b>CONCLUSION</b>JYBDG could decrease the intrauterine transmission of HCMV and is beneficial to fetal growth and development.</p>

Observation on clinical efficacy of combined therapy of zinc supplement and jinye baidu granule in treating human cytomegalovirus infection / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To explore the clinical efficacy of the combined therapy of zinc supplement and Jinye Baidu granule (JBG) on human cytomegalovirus (HCMV) infection.</p><p><b>METHODS</b>One hundred and forty patients with positive HCMV-IgM were randomly divided into four groups, with 35 cases in each group, that is, the control group (only medicated with JBG), the high-, moderate- and low-dose zinc combined groups (treated with JBG combined with zinc gluconate tablet at dose of 30 mg, 20 mg, 10 mg every day respectively). The negative conversion rate of HCMV-IgM was observed.</p><p><b>RESULTS</b>Insignificant difference in the negative conversion rate was shown in comparison of the control group with the low dose group (P > 0.05), and in comparison of the high dose with the moderate dose group (P > 0.05); however, the rate was significantly lower in the control group than that in the moderate dose and high dose group (P < 0.05).</p><p><b>CONCLUSION</b>Combined therapy of zinc supplement and JBG can significantly increase the negative conversion rate of HCMV-IgM. The optimal dosage of zinc gluconate tablet was 20 mg once a day.</p>

Experimental study on effect of jinye baidu preparation in inhibiting human cytomegalovirus protein kinase pul 97 / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To study the inhibitory effect of Jinye Baidu Preparation (JBP), a Chinese medicinal preparation, on human cytomegalovirus protein kinase pu197 and to explore its molecular mechanism in treating human cytomegalovirus (HCMV) infection.</p><p><b>METHODS</b>Expression of the HCMV pu197mRNA in infected cells was measured by semi-quantitative RT-PCR before and after intervention of JBP or Ganciclovir (GCV), and effect of the two medicines on the proliferation activity of the infected cells was observed by MTT.</p><p><b>RESULTS</b>Both JBP and GCV showed obvious inhibitory action on HCMV pu197mRNA. They could significantly enhance the proliferation activity of the cells 72 hours after HCMV infection.</p><p><b>CONCLUSION</b>JBP could inhibit the gene expression and duplication of HCMV by inhibiting the gene expression of HCMV protein kinase pu197 to enhance the proliferation activity of the infected cells so as to achieve its anti-virus action.</p>

Relationship between the pathologic changes of human embryo fibroblast cells and expression of late mRNA after human cytomegalovirus infection in vitro / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the relationship between late mRNA and the cytopathic effect(CPE) and ultrastructural features after human cytomegalovirus (HCMV) infection in vitro.</p><p><b>METHODS</b>Human embryo fibroblast cells(HEL) were infected with HCMV AD169 strain. The expression of the HCMV late mRNA was measured by semiquantitative RT-PCR, the cytopathic effect (CPE) and the cell ultrastructure were observed by means of light microscopy and electron microscopy, respectively.</p><p><b>RESULTS</b>The HCMV late mRNA could be detected 12 hours postinfection and increased gradually, but the CPE appeared 48 hours postinfection in HEL cells. The HCMV infected cells exhibited significant mitochondrial enlargement and the number of mitochondrial ridge deletion, the cisternae lumen of endoplasmic reticulum (ER) dilation and vacuolization (at the end age). The mature nucleocapsid could be observed 96 hours postinfection.</p><p><b>CONCLUSION</b>The ultrastructural changes have an intimate correlation with the expression of HCMV late mRNA and play an important role in the life circle of the virus. HCMV late mRNA may serve as a indicator of the clinical effect of treatment in active HCMV infection.</p>

Experimental study on antivirus activity of traditional Chinese medicine / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the difference of anticytomegaloviral activity of three kinds of traditional Chinese medicines which are the injections of Jinye Baidu, Radix Isatidis and Indigowoa in vitro.</p><p><b>METHOD</b>The inhibitory activity of three traditional Chinese medicines against human cytomeglovirus (HCMV AD169) infected human embryo lung fibroblasts (HELF) was observed by cytopathic effect method (CPE) and MTT method in vitro. According their value of A, anticytomegaloviral activity has evaluated.</p><p><b>RESULT</b>Experimental study in vitro showed that the 50% toxicity dose (TD50) of Jinye Detoxifying, Radix Isatidis root and Indigowoa were 20, 10.23, 20.23 g x L(-1) respectively; the 50% inhibitory concertration (IC50) were 5.65, 3, 5.71 g x L(-1) respectively; the therapeutic index (TI) were 3.54, 3.41 and 3.54 respectively. It suggested that three traditional Chinese medicines had anticytomeglovirus activity and their effect increased with their concentration.</p><p><b>CONCLUSION</b>Three traditional Chinese medicines of the parenteral solution of Jinye Detoxifying, Radix Isatidis root and Indigowoa have antiviral activity when they are diluted in 1:200. They are safe and valuable drug for inhibiting cytomeglovirus infection.</p>

Histologic change in human cytomegalovirus-infected explants of first trimester human placenta and expression of human cytomegalovirus gene / 中华妇产科杂志

Objective To observe histologic changes of human cytomegalovirus(hCMV)-infected explants of first trimester human placenta and expression of hCMV gene in the hCMV-infected explants, and investigate the mechanism of intrauterine transmission of hCMV from mother to fetus.Methods The first trimester placenta explants cultures were carried out, and they were infected with hCMV for 10 days. The expression of hCMV immediate early protein(IEP) 72-IEP86 were determined using indirect-immuno fluorescent assay, and in situ hybridization method was used to examine the hCMV late gene (LG)mRNA. For histologic evaluation of morphological changes in villi, transmission electron microscope was used.Results (1) Typical hCMV-induced lesions bearing hCMV IEP72-IEP86 were consistently localized in the trophoblast of covering placenta villi, interstitial cell and vascular endothelia cell 12 hours after infection, and were predominant in cytotrophoblast. (2) Replication of hCMV in placenta explants culture occurred from 12 hours to 24 hours and disappeared since 48 hours after infection with different concentrations of hCMV when examined by in situ hybridization. (3) Tissue integrity and viability of first trimester placenta explants were obtained in culture for 10 days and then explants were infected with different concentrations of hCMV 100 tissue culture infectious doses(TCID_ 50 ),200 TCID_ 50 and 300 TCID_ 50 , the progression of the infection was observed in the tissue that maintained its normal cellular organization under light microscope. But typical inflammation of cellular organization was observed under transmission electron microscope. Conclusions (1) A flash replication of hCMV in placental explants culture occurs; IEP72-IEP86 may be in intrauterine infection of hCMV for a long time. (2) There are pathological ultrastructure changes in hCMV-infected explants.