Cloning and expression analysis of ANR genes from different species of Lonicera japonica Thunb / 药学学报

Anthocyanidin reductase (ANR) is one of the key enzyme in the flavonoid biosynthetic pathway, and its catalytic activity is important for the synthesis of plant anthocyanin. In this study, specific primers were designed according to the transcriptome data of Lonicera japonica Thunb., and the CDS, gDNA and promoter sequences of ANR genes from Lonicera japonica Thunb. and Lonicera japonica Thunb. var. chinensis (Wats.) Bak. were cloned. The results showed that the CDS sequences of LjANR and rLjANR were 1 002 bp, the gDNA sequences were 2 017 and 2 026 bp respectively, and the promoter sequences were 1 170 and 1 164 bp respectively. LjANR and rLjANR both contain 6 exons and 5 introns, which have the same length of exons and large differences in introns. The promoter sequences both contain a large number of light response, hormone response and abiotic stress response elements. Bioinformatics analysis showed that both LjANR and rLjANR encoded 333 amino acids and were predicted to be stable hydrophobic proteins without transmembrane segments and signal peptides. The secondary structures of LjANR and rLjANR were predicted to be mainly consisted of α-helix and random coil. Sequence alignment and phylogenetic analysis showed that LjANR and rLjANR had high homology with Actinidia chinensis var. chinensis, Camellia sinensis and Camellia oleifera, and were closely related to them. The expression levels of LjANR and rLjANR were the highest in flower buds and the lowest in roots. The expression patterns at different flowering stages were similar, with higher expression levels in S1 and S2 stages and then gradually decreased until reaching the lowest level in S4 stage, after a slow increase in S5 stage, the expression levels decreased again. The expression levels of ANR genes in the two varieties showed significant differences in roots, S2 and S5 stages, while the differences in stems, flower buds, S1, S3 and S6 stages were extremely significant. The prokaryotic expression vector pET-32a-LjANR was constructed for protein expression. The target protein was successfully expressed of about 59 kD. This study lays a foundation for further study on the function of ANR gene and provides theoretical guidance for breeding new varieties of Lonicera japonica Thunb.

Identification and characterization of flavonoid 3-O-glycosyltransferase gene CtUF3GT from safflower (Carthamus tinctorius L.) / 药学学报

UDP-glucose: flavonoid 3-O-glucosyltransferase (UF3GT) uses flavones, dihydroflavonol or anthocyanin as the acceptor and uridine 5′-diphosphate-sugar as the donor to catalyze the production of flavonoid 3-O-glycoside compounds. Based on sequence homology and transcriptome data, we screened and cloned a UF3GT gene named CtUF3GT (GenBank No. OM948976) from safflower. Biological information analysis demonstrate that CtUF3GT has highly conserved PSPG motif. The open reading frame of CtUF3GT is 1 446 bp, encoding 481 amino acids, with a presumed molecular weight of 52.36 kD and a theoretical isoelectric point of 5.33. Multiple sequence alignment indicate that CtUF3GT has a high homology with UF3GT from Asteraceae, and phylogenetic analysis showed that CtUF3GT clusters with functional identified UF3GTs from other species. The purified recombinant protein glucosylated kaempferol and quercetin to biosynthesis of kaempferol 3-O-glucoside and quercetin 3-O-glucoside, respectively. And CtUF3GT prefered to use kaempferol as substrate. qRT-PCR analysis showed that the UF3GT gene was most highly expressed in flowers, followed by leaves, with very low expression in bracts and stems, and no expression in roots. The expression of UF3GT gene showed a trend of increasing and then decreasing at different stages of flower development. The expression of CtUF3GT gene in safflower with different flower color was highly significant (P < 0.01) at S1, S2, S5, S6 and S7 stages of flower development, in which the expression of CtUF3GT in white safflower was 5.3 and 3.1 times higher than that in red safflower at S6 and S7 stages. This study lays the foundation for further exploring the role of CtUF3GT in the mechanism of safflower flavonoid secondary metabolite biosynthesis and accumulation.

The mechanism of methyl jasmonate-induced accumulation of hydroxysafflor yellow A in safflower of different colors / 药学学报

To explore the mechanism hydroxysafflor yellow A (HSYA) biosynthesis and regulation, the effect of methyl jasmonate (MeJA) treatment on gene expression related to the biosynthesis of hydroxysafflor yellow A (HSYA) was analyzed, and expression differences in genes involved in HSYA biosynthesis in safflower of different colors was quantified. MeJA at concentrations of 0, 50, 100, and 200 μmol·L-1 was sprayed onto safflower florets to determine the optimal concentration of MeJA. Safflower was treated with 100 μmol·L-1 MeJA and florets were harvested 0, 3, 6, 12 and 24 h after treatment. The content of MeJA was determined by high performance liquid chromatography (HPLC). RNA was extracted from safflower florets treated with 100 μmol·L-1 MeJA for 6 h. The transcription of key genes involved in the biosynthesis of HSYA was quantified by qRT-PCR and differentially expressed genes were identified. The content of HSYA increased after treatment with MeJA, with 100 μmol·L-1 MeJA treatment for 6 h having the greatest effect on HSYA accumulation. qRT-PCR results showed that MeJA could significantly increase the transcription of HSYA biosynthesis genes including PAL2, PAL4, 4CL2, 4CL4, 4CL5, CHS3, CHS4 and CHI2. The content of HSYA differed between safflowers of different colors with a trend of red>orange-yellow>yellow>white. The results of qRT-PCR showed that the expression of CHS1 and CHI2 in red, orange and yellow safflower was significantly higher than that in white safflower. These results indicate that MeJA promotes the accumulation of HSYA by up-regulating the expression of genes involved in the biosynthesis of HSYA such as PAL2, PAL4, 4CL2, 4CL4, 4CL5, CHS3, CHS4 and CHI2, and the variation of HSYA content in safflower of different colors was related to a difference in the level of expression of CHS1 and CHI2.

Occurrence regularity and integrated control of leaf miner in safflower / 中国中药杂志

Leaf miner is one of the major pests on safflower, which causes yield loss and poor quality seriously. "Weihonghua", "nine safflower varieties" and "three chemical insecticides" as materials that used to evaluate variety and regularity of leaf miner, safflower resistant level, and different proportions insecticides in field efficiency test. The results showed that Liriomyza sativae and L. huidobrensis accounted for 80%, the peak period of two pests was all in July; but Phytomyza horticola is relative less, its peak period occured in June. Three were great difference of resistance to leaf miner among safflower varieties, FQ12 and YJ65 expressed higher resistibility to leaf miner by ratio method. With abamectin 2% emulsifiable concentrate diluted for 2 000 times, or the mixture three insecticides(bifenthrin 20% water emulsions, thiamethoxam 25% water dispersible granule, abamectin 2% emulsifiable concentrate=1∶1∶1) diluted for 3 000 times, which were sprayed on leaves at squaring stage and lethal rate was 96% after 48 h in the study. Through comparative study on the variety and regularity of leaf miner, screen for resistant varieties to leaf miner and for high efficiency pesticide. The study provides theoretical basis and reference for integrated pest management of leaf miner.

Research on regional distribution and industrial status analysis of genuine medicinal resources of flowers in Henan province / 中国中药杂志

Flower medicinal materials usually refer to Chinese medicinal materials with a complete flower,inflorescence,or part of a flower as the different medicinal parts,they have an important share in the Chinese herbal medicine market and appeared frequently in Chinese medicine prescriptions. Firstly,the species and regional distribution of the flower medicinal materials resources in China were briefly summarized. Secondly,the characteristics,yield,producing area and origin distribution of the main flower medicinal materials in Henan province were discussed. Finally,the present situation and the main problems of the flower medicinal materials industry in Henan province were comprehensively analyzed,and the corresponding industrial development countermeasures were put forward.This research was intended to provide decision-making demonstration and scientific basis for the rational exploitation and utilization of resources,breeding of new varieties,planting division,production layout and the healthy and sustainable development of the flower medicinal materials industry in Henan province.

Changes in TGF-beta1/Smads signaling pathway in rats with chemical hepatocarcinogenesis / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the changes in transforming growth factor beta 1 (TGF-beta1)/Smads signaling pathway in rats with chemical hepatocarcinogenesis.</p><p><b>METHODS</b>Fresh diethylnitrosamine (DENA) solution was administered in SD rats to induce hepatocellular carcinoma (HCC). The protein expressions of TGF-beta1, phosphorylated Smad2, Smad4 and Smad7 were detected in these rats with immunohistochemistry, and the mRNA expression of Smad4 was evaluated with RT-PCR.</p><p><b>RESULTS</b>Cirrhotic nodules occurred in the rats 8 weeks after DENA treatment, and HCC nodules were found 16 weeks after the treatment. In the normal liver tissue, very low levels of TGF-beta1 and Smad4 expressions, low Smad7 expression and high phosphorylated Smad2 expression were detected. The development of liver cirrhosis was accompanied by increased expressions of TGF-beta1, Smad4 and Smad7 but at 8 weeks after DENA treatment, the expression of phosphorylated Smad2 was significantly decreased, followed then by gradual increment till nearly the normal level. Twenty-two weeks after DENA treatment, Smad4 expression in liver tissue decreased markedly as compared with the levels at 8 and 16 weeks. The expressions of Smad4 and phosphorylated Smad2 in the HCC tissue was significantly lower than those in normal liver tissue.</p><p><b>CONCLUSION</b>Hepatocarcinogenesis involves very complex mechanisms, can can be related partially to the decreased Smad4 and phosphorylated Smad2 expression and TGFbeta1 and Smad7 overexpression in advanced stage of liver cirrhosis.</p>

Suppression of osteosarcoma in vitro by coexpression of antisense VEGF165 cDNA and thymidine kinase gene / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the effect of VEGF expression in osteosarcoma cell line and the target killing effect of HSV1-TK/GCV system on transfected osteosarcoma cells under hypoxia conditions.</p><p><b>METHODS</b>Eukaryotic expression plasmid with HRE promoter was constructed to express the antisense VEGF165 cDNA and Hygromycin phospho-transferase-thymidine kinase (HyTK) fusion gene. The recombinant vectors were then transfected into osteosarcoma cell line MG63 with lipofectin mediated gene transfer methods. PCR and RT-PCR were used to confirm the presence and expression of TK gene. The sensitivity of transfected cells to GCV and "bystander effect (BSE)" of HSV1-TK/GCV system under normoxia or hypoxia conditions were measured by MTT assay and mixed co-culture experiment. The expression of VEGF protein was detected by ELISA under hypoxia condition. Cell cycle phase distribution was determined by flow cytometry. In addition, electromicroscopy was used to document ultrastructural alterations.</p><p><b>RESULTS</b>The eukaryotic expression vector pBI-HRE-AsVEGF165 -HyTK was constructed successfully. The transfected cell line MG63TV was established and confirmed by PCR and RT-PCR of the presence of transgene and its mRNA expression. GCV was toxic to transfected cells in a concentration-dependent manner. The sensitivity to GCV toxicity was 100 times higher under hypoxia condition than that under normoxic condition. The mixed culture experiments showed that the "bystander effect" was enhanced significantly under hypoxia condition. VEGF expression of transgene cells under hypoxia condition decreased 50% compared to that of normal condition. Under hypoxia and GCV, DNA synthesis of MG63TV cells was inhibited along with an increase of cells at G0 approximately G1 phase, apoptosis and necrosis.</p><p><b>CONCLUSIONS</b>Antisense VEGF expression driven by HRE promoter in combination with hypoxia can provide a target inhibition of VEGF expression in human osteosarcoma cells, with an enhanced selective killing effect and BSE of the HSV-TK/GCV system. The double-gene co-expression system in study provides experimental basis for therapy against osteosarcoma by a synchronous antiangiogenic and suicide gene approach.</p>

Study on the regular pattern of the distribution of skin epidermal stem cells in the different parts of a healthy human body / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the regular pattern of the distribution of skin epidermal stem cells (ESCs) in the different parts of a healthy human body, and to evaluate the feasibility of the identification of ESCs by P63 and CD29 with single and double labeling.</p><p><b>METHODS</b>Full-thickness skin samples from 21 parts (including scalp, dorsum of foot, sole of foot, pubic region, and scrotum) of 5 healthy persons were harvested for the study. Immunohistochemistry method with biotin-streptavidin-horseradish peroxidase (SP) was employed with P63 and CD29 as the first antibody to carry out single and double labeling. The staining results were subjected to image analysis. The distribution of the ESCs in the skin from the above parts was observed and expressed as positive unit (PU) value.</p><p><b>RESULTS</b>It was found by P63 single labeling and P63 and CD29 double labeling that the PU value in the dorsum of foot was the lowest while that in the scalp was the highest among all the parts of a healthy body. It was also found by CD29 single labeling that the PU value in the dorsum of foot was the lowest [(11.9 +/- 1.5)%] while highest in the scalp [(29.1 +/- 5.0)%]. The PU value in the hairy region of a human body was evidently higher than that in the non-hairy region (P < 0.01), when examined by P63 and CD29 single and double labeling. But there was no difference in the PU values between the trunk and limbs by means of P63 and CD29 single and double labeling (P > 0.05).</p><p><b>CONCLUSION</b>There are more ESCs in the skin from the scalp, mons pubis and scrotum than other parts of the body. Single P63 or CD29 labeling exhibits higher sensitivity but lower specificity in the identification of ESCs. While the double labeling method exhibits higher specificity but lower sensitivity. Above all, it seems that the double labeling may be a simple and effective method for the identification of ESCs.</p>

Expression of hepatocyte growth factor/c-Met system in nasopharyngeal carcinoma and its biological significance / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the expression of hepatocyte growth factor (HGF), and its receptor c-Met protein in nasopharyngeal carcinoma (NPC) and CNE-2 NPC cell line, to correlate their expression level with clinicopathologic features and to study the effect of HGF/c-Met system on the invasive and metastatic potential of NPC.</p><p><b>METHODS</b>Forty-five biopsies were collected from pre-treatment NPC patients during the period from 1999 to 2003. Immunohistochemical staining was used to detect the expression of HGF-alpha subunit and c-Met protein in NPC tissues. The association between expression of these proteins and clinicopathologic features was statistically analyzed. The expression of HGF and c-Met, as detected by flow cytometry, in CNE-2 NPC cell line (with or without exogenous HGF) was compared. Western blot analysis and reverse transcriptase-polymerase chain reaction (RT-PCR) were also applied to evaluate the protein and mRNA expression of c-Met in CNE-2 cells.</p><p><b>RESULTS</b>In the 45 cases studied, the expression rate of c-Met was 91.1% (41/45). Only 1 case (2.2%, 1/45) showed positive signal for HGF in neoplastic cells. Instead, HGF was expressed in surrounding lymphocytes. The expression of c-Met positively correlated with lymph node metastasis (P = 0.024). There was also a positive correlation between expression of c-Met by tumor cells and expression of HGF by surrounding lymphocytes (r(s) = 0.450, P = 0.002). Moreover, the expression of c-Met was higher if there was a higher expression of HGF by lymphocytes (P = 0.009). However, there was no association between expression of c-Met and clinicopathologic features, such as age, gender, histopathologic type and clinical stage. After treatment with HGF for 24 hours, the percentage of c-Met-positive cells was significantly increased in CNE-2 cell line, from (46.6 +/- 9.02)% to (85.8 +/- 6.05)% (P = 0.003). The c-Met protein expression and c-Met mRNA level were also enhanced in CNE-2 cells with HGF treatment. However, endogenous HGF was not detected in CNE-2 cells, regardless of HGF treatment.</p><p><b>CONCLUSIONS</b>HGF may play an important role in the development of NPC metastasis by inducing the expression of c-Met in tumor cells via a paracrine, instead of an autocrine, pathway.</p>

The role of CD44 in the proliferation, adhesiveness and invasiveness of osteosarcoma cell lines / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the influence of CD44 a cell-matrix adhesion molecule on the proliferation, adhesiveness and invasiveness of osteosarcoma cell lines, in order to investigate the growth and invasion mechanism of osteosarcoma.</p><p><b>METHODS</b>Three osteosarcoma cell lines MG-63, HOS and U2-OS were routinely cultured. Flow cytometry and Western blot analysis were used for detecting the positive rates and relative amount of CD44 protein in the three cell lines. RT-PCR method was also used to compare the differences in the expression of CD44 mRNA among the 3 cell lines. Then, MTT method, adhesion detection, and Microcon-migration assay were used to detect the changes of the cells' proliferation rate, adhesive and invasive abilities after blocking the role of CD44 by using a special neutralizing antibody.</p><p><b>RESULTS</b>The results of flow cytometry showed that the percentage of CD44 positive cells were both over 99% in HOS and U2-OS, while that in MG-63 was only (2.10 +/- 0.46)%. The average fluorescence density of CD44 in HOS was significantly higher than in U2-OS. Western blot also showed that the relative content of CD44 protein in HOS was notably higher than that in U2-OS, while CD44 was negatively expressed in MG-63. The expression of CD44 mRNA was significantly lower in MG-63 than in both HOS and U2-OS, which were consistent with the expression of CD44 protein. The proliferation rates and adhesive abilities of MG-63 and HOS have no significant difference, but both were significantly higher than that of U2-OS. The invasive abilities of HOS was dramatically higher than MG-63 and U2-OS. After the role of CD44 was blocked by anti-CD44 neutralizing antibody, the proliferation rates of the 3 cell lines did not change significantly. While the HOS and MG-63 adhesion indices decreased dramatically (P < 0.05), the invasive abilities of HOS and U2-OS also decreased notably (P < 0.01).</p><p><b>CONCLUSIONS</b>CD44 could promote the adhesiveness and invasiveness of osteosarcoma cell line HOS. CD44 may take part in promoting the process of U2-OS invasion and the adhesion of MG-63. On the other hand, CD44 could not affect the osteosarcoma cell proliferation rates.</p>

Targeted inhibition of vascular endothelial growth factor (VEGF) expression in human osteosarcoma cell line by antisense VEGF165 cDNA promoted by hypoxia reaction element / 中华病理学杂志

<p><b>OBJECTIVE</b>Utilizing the hypoxia inducible factor 1/hypoxia reaction element (HIF-1/ HRE) gene regulation system to construct antisense vascular endothelial growth factor (VEGF165) cDNA eukaryotic expression vector promoted by HRE, and investigate its targeted inhibiting VEGF expression of osteosarcoma cells in hypoxia environment.</p><p><b>METHODS</b>Eukaryotic expression plasmid with HRE promoter was constructed containing luciferase reporter gene and antisense VEGF165 cDNA by using PCR and recombinant DNA techniques. The recombinant vectors were transfected into osteosarcoma cells with lipofectin method. Hypoxia-inducible reporter gene expression was determined by liquid scintillation analyzer and the expression of VEGF protein was detected by ELISA method.</p><p><b>RESULTS</b>The eukaryotic expression plasmid containing antisense VEGF165 and luciferase promoted by HRE was constructed successfully. After being transferred into MG63 cells, luciferase expression was increased 3.5 x 10(2) times and VEGF protein expression decreased 45% under hypoxia condition.</p><p><b>CONCLUSION</b>Antisense VEGF165 cDNA expression, efficiently realized by HRE promoter under hypoxia condition, provides an experimental basis for targeted antiangiogenesis of tumors.</p>

The study of inhibition effect of octreotide on the growth of hepatocellular carcinoma xenografts in situ in nude mice / 中华外科杂志

<p><b>OBJECTIVE</b>To observe the effect of octreotide (OCT) on inhibiting hepatocellular carcinoma (HCC) and investigate its mechanisms.</p><p><b>METHODS</b>Nude mice bearing xenografts in situ were treated with OCT or saline control for 7 weeks since tumor implantation. The immunohistochemistry for somatostatin receptor 2 (SSTR2), cMet, transforming growth factor beta1 (TGFbeta1), phospho-Smad2, Smad4 and Smad7 was performed. SSTR2 and Smad4 mRNA expression was measured by semi-quantitative RT-PCR.</p><p><b>RESULTS</b>After OCT treatment, the mean tumor weight in mice given OCT (0.17 +/- 0.14 g) was significantly lower than that of the control group (0.53 +/- 0.06 g). The inhibition rate of tumor was 67.9%. mRNA and protein expression of SSTR2, Smad4 in tumor cells of the treatment group were significantly more than that of the control group. cMet expression in OCT group was remarkably lower than that in control group. Between two groups, the expression of TGFbeta1, phospho-Smad2 and Smad7 were not remarkably different. In addition, phospho-Smad2 expression in HCC was significantly less than that of the normal hepatic cell.</p><p><b>CONCLUSION</b>OCT can inhibit the growth of HCC xenografts markedly. The mechanisms of OCT-induced inhibition effect may be related to up-regulating SSTR2 expression, down-regulating cMet, and recovering the function of TGFbeta/Smads-induced antitumor. In addition, the decreased expression of phospho-Smad2 may be an important feature of Bel7402 cells.</p>

Influence of aerosols on the expression of cyclin B1, cyclin C and proliferating cell nuclear antigen in wound tissue healing of burned rat models / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate the influence of aerosols on the expression of cyclin B(1), cyclin C and proliferating cell nuclear antigen (PCNA) in wound tissue healing of burned rat models.</p><p><b>METHODS</b>Sprague Dawley (SD) rats were inflicted as the deep partial thickness burn models. Rats were randomly divided into experimental group and control group. The experimental group were treated with aerosols. Samples were collected in 1 approximately 10 postburn days. Immunohistochemistry and image analysis methods were conducted to examine the expression of cyclin B(1), cyclin C and PCNA in both experimental and control groups.</p><p><b>RESULTS</b>The expression of cyclin C in experimental group was detected in nucleus of skin basal cell on the second postburn day, increased evidently at the fifth days and sustained at high expression level up to the tenth days after treatment. The expression of cyclin C in experimental group was significantly higher than control group (P < 0.05). The expression of PCNA was first observed in skin basal cell nucleus and hair follicle cell nucleus in both experimental and control group on the third postburn day. The expression of PCNA increased evidently at the fifth days in experimental after treatment and that increased evidently at the seventh days in control group, which showed there were lots of active proliferation cell. And the difference of the expression of PCNA between experimental and control group was significant (P < 0.01). The expression of cyclin B(1) was detected in nucleus and cytoplasm of skin basal cell in both groups on the third postburn day, and no difference between the experimental and control group (P > 0.05).</p><p><b>CONCLUSIONS</b>Aerosols can up-regulate the expression of cyclin C and PCNA in skin basal cell nucleus. Therefore the aerosols can accelerate wound tissue healing.</p>

Effect of survivin antisense mRNA transfection on the growth and chemotherapy sensitivity of lymphoma cells / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the effect of transfecting survivin antisense mRNA on growth and chemotherapy sensitivity of lymphoma cells.</p><p><b>METHODS</b>Eukaryotic expression plasmid pcDNA3. 1-antisense (As) survivin was constructed and transfected into Jurkat T lymphoblastic lymphoma cell lines with high expression survivin mRNA by use of lipofectmine gene transfer technique. Expression of survivin mRNA and protein were detected by reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemical and Western blot. The effect of transfecting survivin antisense mRNA on the growth of Jurkat cell lines was monitored by population doubling time (PDT) and Apoptotic indexes (AI). The morphologic features were observed in transfected cells by light and electric microscopes. MTT assay was used to analyze the response of transfected cells to CTX and MTX.</p><p><b>RESULTS</b>Compared with the control cells, the expression of survivin mRNA and protein were reduced after transfected pcDNA3. 1-Assurvivin 48 h, 5 w and 6 w, PDT (52 h) was prolonged. Apoptotic indexes were higher in transfected antisense survivin mRNA cells [20.2% (48 h)], 6.2% (5 w) and 6.8% (6 w) than control ones [2.1%, 1.3% (48 h)] and [1.3% (5 w) and 1.0% (6 w)]. The cells grow slowly and the dead cells increase and some swelling and apoptotic cells were observed in transfected pcDNA3. 1-Assurvivin groups by invert, light and electric microscopes. The Jurkat cell line of transfected pcDNA3. 1-Assurvivin had higher sensitivity to CTX and MTX. The rate of inhibition was higher in transfected group. There is a significant difference between the transfected group and untransfected one, P < 0.05.</p><p><b>CONCLUSIONS</b>The result indicated that survivin gene was very important for growth of Jurkat cells. To inhibit the expression of survivin will be significant in therapy of T lymphoblastic lymphoma. Survivin gene might be a target of therapy.</p>

Macrophage migration inhibitory factor enhances neoplastic cell invasion by inducing the expression of matrix metalloproteinase 9 and interleukin-8 in nasopharyngeal carcinoma cell lines / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Nasopharyngeal carcinoma (NPC) shows highly invasive and metastatic features. This study aims to investigate macrophage migration inhibitory factor (MIF)-induced invasion of NPC cells in vitro and the effects on matrix metalloproteinases (MMPs) and interleukin-8 (IL-8), and to study the mechanism of tumor cell invasion and metastasis in the early stage of NPC.</p><p><b>METHODS</b>Two nasopharyngeal carcinoma cell lines, CNE-1 and CNE-2, were adopted in this study. The NPC cell invasion and migration were evaluated by microinvasion assay. The variation of expression percentages of MMP2- or MMP9-positive cells was detected by flow cytometry in two cell lines with or without MIF treatment. Western blotting and RT-PCR were used to assay the protein and mRNA expressions of MMP2 and MMP9. The IL-8 concentration secreted by NPC cells was compared with the cells with different treatments using ELISA.</p><p><b>RESULTS</b>After treating with MIF for 48 hours, the cell numbers of CNE-1 and CNE-2 which went through the 8-microm filter membrane were increased. Compared with non-MIF treated NPC cells, significant difference could be found both in CNE-1 (P = 0.005) and CNE-2 cells (P = 0.001). The percentages of MMP9-positive cells were significantly increased in both CNE-1 [from (28.5 +/- 2.5)% to (82.4 +/- 3.5)%, P = 0.001] and CNE-2 [from (32.8 +/- 3.5)% to (86.1 +/- 1.6)%, P = 0.002]. The relative intensity of MMP9 protein expression was also enhanced in both cell lines (CNE-1: from 83.1 +/- 6.0 to 242.9 +/- 22.9, P = 0.002; CNE-2: from 84.4 +/- 4.3 to 278.9 +/- 29.7, P = 0.003). Correspondingly, the increased MMP9 mRNA expression level was significantly detectable in both cell lines. The concentration of IL-8 in the supernatant of CNE-2 was higher [(1201.8 +/- 593.3) pg/ml] after treatment. It was also remarkably higher than that in the supernatant of CNE-2 without treatment (P = 0.026). However, there was no significant difference in the concentration variation of IL-8 in CNE-1 (P = 0.581), while the IL-8 mRNA level was only enhanced in CNE-2.</p><p><b>CONCLUSIONS</b>MIF can induce potent invasion of NPC cell lines in vitro, and the infiltrating lymphocytes in NPC might be responsible for the invasion and metastasis of tumor cells. MIF cytokine which is secreted by these infiltrating lymphocytes might contribute to the invasion as well as metastasis of NPC in the early stages by induction of MMP9 and IL-8 in an indirect pathway.</p>

Association of E-cadherin and beta-catenin with metastasis in nasopharyngeal carcinoma / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>This study was designed to detect methylation of E-cadherin gene promoter and gene mutation of beta-catenin in exon 3 and their expression of protein and mRNA in primary tumor and lymph node metastatic tumor of nasopharyngeal carcinoma (NPC), and investigate the mechanism of invasion and metastasis of neoplastic cells in NPC.</p><p><b>METHODS</b>Fourty-two fresh biopsy samples were taken from untreated NPC patients at the Affiliated Hospital of Sun Yat-sen Medical College, Sun Yat-sen University, Guangzhou, China during the period of 1999-2002. Among them 21 were taken from primary tumors and the other 21 from lymph node metastatic tumors. The gene promoter methylation of E-cadherin was detected by methylation-specific PCR (MSP). The mutation in exon 3 of beta-catenin was detected by direct sequencing analysis. RT-PCR, Western blot and immunohistochemical staining were used to detect the mRNA and protein expression patterns in both primary and metastatic tumors of NPC.</p><p><b>RESULTS</b>Down-regulated expression of E-cadherin in metastatic tumor was compared with that in primary tumor. Reduced expression of E-cadherin was found to be correlated with lymph node metastatic tumor of NPC (P = 0.004); but there was no obvious correlation between primary and metastatic tumors in the expression of beta-catenin (P = 0.698). The mRNA expression level of E-cadherin in metastatic tumors decreased significantly compared with that in primary tumors. However, little change was observed in the mRNA level of beta-catenin in different tumor tissues. Only 4 samples (19.1%) displayed gene promoter methylation of E-cadherin in primary tumor and 10 samples (47.6%) showed methylated form of E-cadherin. The gene promoter methylation of E-cadherin was more common in metastatic tumor than in primary tumor of NPC (P = 0.024). Only 2 (4.76%) of the 42 samples showed mutations in exon 3 of beta-catenin at 41 (T41A, ACC-->GCC) and codon 47 (S47T, AGT-->ACT). The cytoplasmic and nuclear expression of beta-catenin in tumor was not found in any samples of NPC.</p><p><b>CONCLUSIONS</b>The results suggest that the downregulation of E-cadherin results from the gene promoter aberrant methylation of E-cadherin and that the methylation of E-cadherin plays an important role in invasion and metastasis of tumor cells in NPC. However, beta-catenin mutation is an infrequent event in NPC, and beta-catenin is not a critical factor influencing the invasion and metastasis of tumor cells in NPC.</p>

A preliminary study on the identification and distribution of epidermal stem cells in different degrees of burn wounds in scalded rats / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the distribution of epidermal stem cells (ESCs) in different degrees of burn wounds in scalded rats.</p><p><b>METHODS</b>Thirty-two Sprague-Dawley (SD) rats were employed in the study. First degree (I), shallow (shallow II) and deep partial thickness (deep II) and full thickness burn wounds (III) were created on the rat skin. Burn wound samples were harvested at 24 postburn hours (PBHs) from all the wounds and were processed to tissue slices. The tissue slices were stained by immunohistochemistry technique. The expression and distribution of ESCs in different degrees of burn wounds were observed with integrins alpha 2 beta 1 and keratin 10 (K10) as first antibodies.</p><p><b>RESULTS</b>K10 positive cells were found to distribute in the strata spinosum, granulosum and lucidum in the first degree burn wound (I) with large amounts of integrins alpha 2 beta 1 positive cells in the residual basal layer and skin appendages (hair follicles) in shallow partial thickness burn wound (shallow II degree), and there were less integrins alpha 2 beta 1 positive cells in the remaining skin appendages in deep dermis in deep partial thickness burn wound (deep II degree). Finally, integrins alpha 2 beta 1 positive cells were sparsely found in the III degree burn wound.</p><p><b>CONCLUSION</b>The distribution of ESCs in burn wounds was closely related to the depth of burn wound. The residual ESCs might be the origin of burn wound regeneration and reepithelization.</p>

A research of endothelial cell-targeted therapy for cure of hypertrophic scar / 中华整形外科杂志

<p><b>OBJECTIVE</b>To investigate the feasibility of endothelial cell-targeted therapy to cure post-burn hypertrophic scar.</p><p><b>METHODS</b>A hypertrophic scar animal model was made. Intralesional injecting of VEGF monoclonal antibody was performed for three weeks. The changes of scar in volume and morphology were observed.</p><p><b>RESULTS</b>1. The volume of scar decreased. 2. The number of the capillary, the amount of collagen I and collagen III decreased. 3. Transmission electron microscope examinations demonstrated many dead or apoptotic fibroblasts and endothelial cells. Fibrocytes were seen relatively common.</p><p><b>CONCLUSION</b>VEGF induces the growth and development of hypertrophic scar in that it induces excessive and uncontrollable angiogenesis, which favors excessive collagen synthesis. Endothelial cell-targeted therapy may be a promising method to cure post-burn hypertrophic scar.</p>