Contamination status and exposure risk of mycotoxins in Coicis Semen / 中国中药杂志

By investigating the contamination status and predicting the exposure risk of mycotoxin in Coicis Semen, we aim to provide guidance for the safety supervision of Chinese medicinal materials and the formulation(revision) of mycotoxin limit standards. The content of 14 mycotoxins in the 100 Coicis Semen samples collected from five major markets of Chinese medicinal materials in China was determined by UPLC-MS/MS. The probability evaluation model based on Monte Carlo simulation method was established after Chi-square test and One-way ANOVA of the sample contamination data. Health risk assessment was performed on the basis of margin of exposure(MOE) and margin of safety(MOS). The results showed that zearalenone(ZEN), aflatoxin B_1(AFB_1), deoxynivalenol(DON), sterigmatocystin(ST), and aflatoxin B_2(AFB_2) in the Coicis Semen samples had the detection rates of 84%, 75%, 36%, 19%, and 18%, and the mean contamination levels of 117.42, 4.78, 61.16, 6.61, and 2.13 μg·kg~(-1), respectively. According to the limit standards in the Chinese Pharmacopoeia(2020 edition), AFB_1, AFs and ZEN exceeded the standards to certain extents, with the over-standard rates of 12.0%, 9.0%, and 6.0%, respectively. The exposure risks of Coicis Semen to AFB_1, AFB2, ST, DON, and ZEN were low, while 86% of the samples were contaminated with two or more toxins, which needs more attention. It is suggested that the research on the combined toxicity of different mycotoxins should be strengthened to accelerate the cumulative exposure assessment of mixed contaminations and the formulation(revision) of toxin limit standards.

Utilization of used fungus-growing materials of Gastrodia elata / 中国中药杂志

This study aims to explore the resource utilization of used fungus-growing materials produced in the cultivation of Gastrodia elata. To be specific, based on the production practice, this study investigated the recycling mechanism of used fungus-growing materials of G. elata by Phallus inpudicus. To screen edible fungi with wide adaptability, this study examined the allelopathic effects of Armillaria mellea secretions on P. impudicus and 6 kinds of large edible fungi and the activities of enzymes related to degradation of the used fungus-growing materials of G. elata. The results showed that P. impudicus can effectively degrade cellulose, hemicellulose, and lignin in used fungus-growing materials of G. elata. The cellulase activity of A. mellea was significantly higher than that of P. impudicus, and the activities of lignin peroxidase, polyphenol oxidase, and xylanase of P. impudicus were significantly higher than those of A. mellea, which was the important reason why A. mellea and P. impudicus used different parts and components of the used fungus-growing materials to absorb carbon sources and develop ecological niche differences. The growth of P. impudicus was significantly inhibited on the used fungus-growing materials of G. elata. The secretions of A. mellea had allelopathic effects on P. impudicus and other edible fungi, and the allelopathic effects were related to the concentration of allelopathy substances. The screening result showed that the growth and development of L. edodes and A. auricular were not significantly affected by 30% of A. mellea liquid, indicating that they had high resistance to the allelopathy of A. mellea. The results showed that the activities of extracellular lignin peroxidase, polyphenol oxidase, and xylanase of the two edible fungi were similar to those of P. impudicus, and the cellulase activity was higher than that of P. impudicus. This experiment can be further verified by small-scale production tests.

Construction of protoplast genetic transformation system for Mycena--symbiont of Gastrodia elata / 中国中药杂志

Mycena, a symbiont of Gastrodia elata, promotes seed germination of G. elata and plays a crucial role in the sexual reproduction of G. elata. However, the lack of genetic transformation system of Mycena blocks the research on the interaction mechanism of the two. In order to establish the protoplast transformation system of Mycena, this study analyzed the protoplast enzymatic hydrolysis system, screened the resistance markers and regeneration medium, and explored the transient transformation. After hydrolysis of Mycena hyphae with complexes enzymes for 8 h and centrifugation at 4 000 r·min~(-1), high-concentration and quality protoplast was obtained. The optimum regeneration medium for Mycena was RMV, and the optimum resistance marker was 50 mg·mL~(-1) hygromycin. The pLH-HygB-HuSHXG-GFP-HdSHXG was transformed into the protoplast of Mycena which then expressed GFP. The established protoplast transformation system of Mycena laid a foundation for analyzing the functional genes of Mycena and the molecular mechanism of the symbiosis of Mycena and G. elata.

Correlation analysis between continuous cropping obstacle of Gastrodia elata and Ilyonectria fungi and relieving strategy / 中国中药杂志

The continuous cropping obstacle of Gastrodia elata is outstanding, but its mechanism is still unclear. In this study, microbial changes in soils after G. elata planting were investigated to explore the mechanism correlated with continuous cropping obstacle. The changes of species and abundance of fungi and bacteria in soils planted with G. elata after 1, 2, and 3 years were compared. The pathogenic fungi that might cause continuous cropping diseases of G. elata were isolated. Finally, the prevention and control measures of soil-borne fungal diseases of G. elata were investigated with the rotation planting pattern of "G. elata-Phallus impudicus". The results showed that G. elata planting resulted in the decrease in bacterial and fungal community stability and the increase in harmful fungus species and abundance in soils. This change was most obvious in the second year after G. elata planting, and the soil microbial community structure could not return to the normal level even if it was left idle for another two years. After G. elata planting in soils, the most significant change was observed in Ilyonectria cyclaminicola. The richness of the Ilyonectria fungus in soils was significantly positively correlated with the incidence of G. elata diseases. When I. cyclaminicola was inoculated in the sterile soil, the rot rate of G. elata was also significantly increased. After planting one crop of G. elata and one to three crops of P. impudicus, the fungus community structure in soils gradually recovered, and the abundance of I. cyclaminicola decreased year by year. Furthermore, the disease rate of G. elata decreased. The results showed that the cultivation of G. elata made the Ilyonectria fungi the dominant flora in soils, and I. cyclaminicola served as the main pathogen of continuous cropping diseases of G. elata, which could be reduced by rotation planting with P. impudicus.

Isolation, identification, and pathogenicity research of brown rot pathogens from Gastrodia elata / 中国中药杂志

Brown rot is a common disease in the cultivation and production of Gastrodia elata, but its pathogens have not been fully revealed. In this study, the pathogenic fungi were isolated and purified from tubers of 77 G. elata samples with brown rot. Pathogens were identified by the pathogenicity test and morphological and molecular identification. The pathogenicity of each pathogen and its inhibitory effects on Armillaria gallica were compared. The results showed that 119 strains of fungi were isolated from tubers of G. elata infected with brown rot. Among them, the frequency of separation of Ilyonectria fungi was as high as 42.01%. The pathogenicity test showed that the pathogenicity characteristics of six strains of fungi were consistent with the natural symptoms of brown rot in G. elata. The morphological and molecular identification results showed that the six strains belonged to I. cyclaminicola and I. robusta in the Nectriaceae family of Sordariomycetes class, respectively. Both types of fungi could produce pigments, conidia, and chlamycospore, and the growth rate of I. cyclaminicola was significantly higher than that of I. robusta. The comparison of pathogenicity showed that the spots formed by I. cyclaminicola inoculation were significantly larger than those of I. robusta inoculation, suggesting I. cyclaminicola was superior to I. robusta in pathogenicity. The results of confrontation culture showed that I. cyclaminicola and I. robusta could signi-ficantly inhibit the germination and cordage growth of A. gallica. A. gallica also inhibited the growth of pathogens, and I. cyclaminicola was less inhibited as compared with I. robusta. The results of this study revealed for the first time that I. cyclaminicola and I. robusta were the pathogens responsible for G. elata brown rot.

Resistance of different ecotypes of Gastrodia elata to tuber rot / 中国中药杂志

Tuber rot has become a serious problem in the large-scale cultivation of Gastrodia elata. In this study, we compared the resistance of different ecotypes of G. elata to tuber rot by field experiments on the basis of the investigation of G. elata diseases. The histological observation and transcriptome analysis were conducted to reveal the resistance differences and the underlying mechanisms among different ecotypes. In the field, G. elata f. glauca had the highest incidence of tuber rot, followed by G. elata f. viridis, and G. elata f. elata and G. elata f. glauca×G. elata f. elata showed the lowest incidence. Tuber rot showcased obvious plant source specificity and mainly occurred in the buds and bottom of G. elata plants. After infection, the pathogen spread hyphae in host cortex cells, which can change the endophytic fungal community structure in the cortex and parenchyma of G. elata. G. elata f. glauca had thinner lytic layer and more sugar lumps in the parenchyma than G. elata f. elata. The transcription of genes involved in immune defense, enzyme synthesis, polysaccharide synthesis, carbohydrate transport and metabolism, hydroxylase activity, and aromatic compound synthesis had significant differences between G. elata f. glauca and G. elata f. elata. These findings suggested that the differences in resis-tance to tuber rot among different ecotypes of G. elata may be related to the varied gene expression patterns and secondary metabolites. This study provides basic data for the prevention and control of tuber rot and the improvement of planting technology for G. elata.

Thoughts and suggestions on ecological cultivation of Gastrodia elata / 中国中药杂志

Due to the special biological characteristics, Gastrodia elata suffers from high resource consumption and low utilization rate in modern agricultural production, which significantly block the green and healthy development of this industry. Based on the theory and technology in ecological cultivation of Chinese medicinal materials, this study analyzed the challenges in ecological cultivation of G. elata, such as waste of fungus material, a few cultivation modes available, continuous cropping obstacles, frequent occurrence of diseases, and poor stability of ecological structure. According to the production practice, the following suggestions were proposed for ecological cultivation of G. elata: following the principle of environmental protection and no pollution, selecting suitable habitats to yield high-quality medicinal materials, committing to green control of diseases and pests, upgrading industrial structure to maximize the benefits, establishing a sound mechanism for protecting the genetic diversity of wild G. elata, carrying out simulative habitat cultivation to improve medicinal material quality, adopting science-based planning of fungus resources to relieve forestry pressure, enhancing the recycling and utilization of fungus materials, and applying diversified cultivation modes to improve the stability of ecological structure. The result is expected to provide a reference for the quality development of G. elata industry.

Methods for synchronous detection of 14 mycotoxins in Pseudostellariae Radix and investigation on its contamination / 中国中药杂志

This study aimed to establish a method for synchronous detection of 14 mycotoxins in Pseudostellariae Radix and investigate its contamination with mycotoxins, so as to provide technical guidance for monitoring the quality of Chinese medicinal materials and medication safety. The sample was extracted with 80% acetonitrile in an oscillator for 1 h, purified using the modified QuEChERS purifying agent(0.1 g PSA + 0.3 g C_(18) + 0.3 g MgSO_4), and separated on a Waters HSS T3 chromatographic column(2.1 mm×100 mm, 1.8 μm). The gradient elution was carried out with 0.1% formic acid in water and acetonitrile, followed by the scanning in the multi-reaction monitoring(MRM) mode and the analysis of mycotoxin contamination in 26 Pseudostellariae Radix samples. The recovery rates of the established method were within the range of 82.17%-113.6%, with the RSD values less than 7% and the limits of quantification(LOQ) being 0.019-0.976 μg·kg~(-1). The detection rate of 14 mycotoxins in 26 batches of medicinal materials was 53.85%. The detection rate of sterigmatocystin(ST) was the highest, followed by those of zearalenone(ZEN), aflatoxin G_2(AFG_2), fumonisin B_1(FB_1), HT-2 toxin, and nivalenol(NIV). Their respective detection rates were 38.46%, 26.92%, 23.08%, 11.54%, 11.54%, and 7.69%, with the pollution ranges being 1.48-69.65, 0.11-31.05, 0.11-0.66, 0.28-0.83, 20.86-42.56, and 0.46-1.84 μg·kg~(-1), respectively. The established method for the detection of 14 mycotoxins is accurate, fast and reliable. The research results have very important practical significance for guiding the monitoring and prevention and control of exogenous fungal contamination of Chinese medicinal materials.

Methyl jasmonate stimulates the synthesis and accumulation of asperosaponin VI in the roots of Dipsacus asper / 药学学报

We investigated the effect of methyl jasmonate (MeJA) on the content of asperosaponin VI and the expression of genes involved in its synthesis. Dipsacus aspero seedlings were treated with MeJA at different concentrations of 50, 100, 150, 200 and 300 μmol·L-1, and leaves and roots were sampled following treatment for 1, 3 and 5 days. The content of asperosaponin VI and superoxide anion in the roots, malondialdehyde (MDA) content in leaves and superoxide dismutase were determined. The results show that 150 μmol·L-1 MeJA significantly increased the accumulation of asperosaponin VI in roots. The content of asperosaponin VI was greatest after treatment for 3 days, and was 2.16 times higher than the control. After MeJA treatment, SOD activity decreased and MDA content increased in leaves. Moreover, superoxide anion content in roots increased. The expression of squalene epoxidase (DaSE1) and geranyl diphosphate synthase (DaGPS), key enzymes in the synthesis of asperosaponin VI, were up-regulated compared with the control group. These results indicate that an optimal concentration of 150 μmol·L-1 MeJA increases the accumulation of asperosaponin VI by up-regulating the expression of key enzymes involved in the synthesis of asperosaponin VI, which facilitates resistance to adversity stress stimulated by MeJA.

Analysis of ecological recycling modes for Gastrodia elata cultivation / 中国中药杂志

Gastrodia elata is a kind of precious traditional Chinese medicine. In artificial cultivation, it has not got rid of its dependence on forest resources. In order to maintain the balance of the ecological system and reduce the waste of resources as much as possible, based on the information from field investigation at many places, this paper introduced the new ecological circulation planting patterns of G. elata, such as "forest-G. elata" supporting planting, G. elata-edible mushroom rotation, forest-G. elata-edible mushroom three-dimensional planting, fungus material classification planting technology, and so on. In this paper, we expounded the ecological problems solved by several planting patterns in G. elata production and analyzed their shortcomings. Finally, based on the exis-ting models, a complete ecological planting system of G. elata was summarized. This planting system emphasizes: ① The follow-up forests should be started before the planting of G. elata. And the economic forests were used to cultivation of G. elata. ② The classified utilization of fungus-growing materials. The leaves were used to cultivate germination bacteria of G. elata, the small branches were used to cultivate protocorm and juvenile tuber, the large branches were used to cultivate immature tuber, and the tree trunk was used to cultivate mature tuber. ③ Recycle utilization G. elata fungus material. The old fungus materials were used to produce strains or cultivate edible fungus. This design project not only solves the problems of the source of G. elata fungus material, the efficient utilization of fungus material and land resources, but also enriches the industrial structure. Using limited time and land resources to obtain greater economic benefits. It has certain guiding significance for poverty alleviation and ecological improvement.

Technical evaluation and principle analysis of simulative habitat cultivation of Dendrobium nobile / 中国中药杂志

The technique of "simulative habitat cultivation" is to preserve the quality of traditional Chinese medicine by simulating the original habitat and site environment of wild Chinese medicine resources. Dendrobium nobile is the most representative variety of traditional Chinese medicine which reflects the coordinated development of medicinal material production and ecological environment. In this paper, the main technical points of the simulated cultivation model of D. nobile were summarized as follows: rapid propagation of seedling tissue technology to ensure the genetic stability of provenance; line card+fermented cow manure+live moss method to improve the survival rate; epiphytic stone cultivation to improve the quality of medicinal materials; and the integration of mycorrhizal fungi to improve the quality stability of medicinal materials. On the basis of summarizing the ecological benefits, economical and social benefits generated by the application of the technology, the paper systematically analyzes the principle of the technology for the cultivation of D. nobile to promote the excellent quality, the light, gas, heat and fertilizer resources of the undergrowth niche are in line with the wild site environment of D. nobile. The rich and complex soil microbial community in the forest laid the foundation for the species diversity needed for the growth of D. nobile.The stress effect on the growth of D. nobile resulted in the accumulation of secondary metabolites. The symbiotic relationship between the symbiotic fungi such as bryophytes and D. nobile promotes the synthesis of plant secondary metabolites. The high quality D. nobile was produced efficiently by improving and optimizing the cultivation techniques.

Isolation and Identification of Mycena of Pleurotus in Wumeng Mountain Area and Optimization of Its Production Conditions / 中国实验方剂学杂志

Objective:Isolate and identify Mycena, expand the resources of geminating fungus of Gastrodia elata and optimize the culture conditions of Mycena，in order to provide information and guidance for the production of geminating fungus of G. elata. Method:Juvenile tuber tissue mass transfer separation and purification technology was used for the separation and purification of strains，traditional morphology microscopy was used to isolate the colony mycelia spores morphological characteristics, such as identification，polymerase chain reaction（PCR） amplification rDNA （Ribosomal DNA） internal transcribed spacer（ITS） was used for sequencing analysis and further homology with NCBI database retrieval，MEGA6 software was used to establish Phylogenetic tree by the Maximum likelihood method （MaximumLikelihood，M-L）, so as to classify and identify isolated strains. At the same time，orthogonal test was used to optimize the optimal growth conditions of Mycena. Result:A total of 86 strains were isolated, which belong to 21 species in 12 genera. WMMFJ，SHXG，WMM-21 and MFJ8103 were identified as M. purpureofusca, and ZT01-6 and ZT01-8 were identified as M. cf. purpureofusca. The growth rate of Mycena in wheat bran medium was significantly higher than in PDA medium. The optimal medium composition for the growth of germinating bacteria was 100 g potato，150 g wheat bran and 20 g corn flour，100 g glucose. And 1，3，5-Trihydroxybenzene significantly promoted the growth of WMMFJ，and played a role in promoting the growth of WMM-21 and ZT01-6，and 2-Methoxyphenol promoted the growth of WMMFJ. Conclusion:Six strains of Mycena were isolated and identified，four of them are M. purpureofusca，and two of them are M. cf. Purpureofusca. The separation method improved the separation effect of germinating bacteria.

Effects of High Temperature Stress on Growth Characteristics of Armillaria / 中国实验方剂学杂志

Objective:To explore the effects of high temperature stress on the growth characteristics of different Armillaria strains，and to provide guidance for screening excellent Armillaria strains with high-temperature resistance. Method:14 strains of Armillaria from different G. elata producing areas were used as experimental materials to observe the growth characteristics and conduct phenotypic classification for the strains. rDNA-IGS sequence analysis was used for molecular identification to further determine the genetic relationship of the tested strains.The strain growth rate, biomass，mycelial length and other indicators under the condition of 23 ℃ （CK） and 30 ℃ high temperature stress were recorded. Result:All the 14 strains of Armillaria had the highest similarity and the closest relationship with Armillaria gallica，but there were significant differences in growth characteristics among different G. elata producing areas. The 14 strains of Armillaria were classified into Ⅳ groups，and the growth status was groupⅠ>group Ⅱ>group Ⅲ>group Ⅳ. After treatment with high temperature stress，the tolerance of each strain to high temperature also showed obvious differences，as shown in the average growth rate of the mycelial was GZ16>SX1>GZ1. The rank of relative mycelial length was GZ16>SX1>GZ3 and the relative biomass was GZ16>SX4>GZ1>HB1>AH2. Conclusion:Under high temperature stress，GZ16 was best in growth rate，relative length of mycelial，relative biomass and growth state，followed by SX1 and GZ1 strains. The results indicate that strains GZ16，SX1 and GZ1 have the strong resistance to high temperature and excellent growth characteristics at normal temperature，so these three strains are suitable to be produced in main G. elata producing areas in China.

Association Study Between Rhizoma Gastrodia elata Brown-rot and Soil Microbial Communities / 中国实验方剂学杂志

Objective:To study the microbial community composition and diversity of brown-rot Gastrodia elata and its surface soil，in order to explain the relationship between brown-rot G. elata and soil microflora in G. elata planting process and provide theoretical basis for revealing the reasons of G. elata brown-rot disease. Method:Used internal transcribed spacer region（ITS） and 16S rDNA high-throughput sequencing technologies to detect the microbial diversity，community structure composition and community structure similarity of fungi and bacteria in healthy tuber，Brown-rot tuber，healthy soil and Brown-rot soil. Result:Compared with health groups，the number of fungi and bacteria operational taxonomic units（OTUs） was increased in brown-rot G. elata and its soil， and the abundance and diversity of fungi and bacteria in brown-rot G. elata soil were significantly decreased. The diversity of fungi in the tubers of brown-rot G. elata was significantly reduced，while the diversity of bacteria was significantly decreased. At the genus level， Mortierella was dominant fungi genus in healthy tuber and healthy soil，which was reduced 7.62% and 15.75% respectively in brown-rot tuber and brown-rot soil. And the dominant bacteria genus was Bradyrhizobium and Burkholderia-Paraburkholderia respectively. Ilyonectria was dominant fungi genus in brown-rot tuber and brown-rot soil，the dominant bacteria genus was Serratia and Bradyrhizobium respectively. Conclusion:The fungal flora in the tuber of brown-rot G. elata had a very high degree of similarity to that in the surrounding soil. These results indicated that the change of soil microbial fungal community caused the occurrence of G. elata brown-rot disease to a certain extent. And the pathogenic fungal Ilyonectria was dominant genus in fungi community of brown-rot tuber and brown-rot soil. Ilyonectria may have the main G. elata brown-rot disease pathogen.

PAR-1 regulation of intracellular Ca²(+) mobilization in pulmonary giant cell carcinoma cell line PLA801D/PLA801C / 中华病理学杂志

<p><b>OBJECTIVES</b>To investigate molecular mechanisms of PAR-1 regulation on intracellular Ca²(+) mobilization in lung giant cell carcinoma cells in vitro and its involvement in tumor metastasis.</p><p><b>METHODS</b>Free intracellular Ca²(+) ([Ca²(+)]i) was measured in lung giant cell carcinoma PLA801C and PLA801D cells by confocal microscopy. Sense and anti-sense PAR-1 expression vectors were transfected into PLA801C (C+)and PLA801D(D-) cells, respectively. The effects of PAR-1 expression were investigated by thrombin and TRAP-induced mobilization of [Ca²(+)]i in the C+ and D-cells.</p><p><b>RESULTS</b>There were significant differences of the mean values of [Ca²(+)]i between PLA801D (59.55) and PLA801C cells (35.46, P < 0.01). The mean [Ca²(+)]i of C+ cells (45.77) was significantly higher than that of its control CV cells (35.46, P < 0.05), and the mean [Ca²(+)]i of D-cells (48.42) was significantly lower than that of its control DV cells (59.55, P < 0.05). The peaks of [Ca²(+)]i of C+ and CV cells were 48.19 ± 9.84 and 45.64 ± 9.87 (P < 0.05) respectively at 80 s and 100 s after thrombin treatment, but were 111.31 ± 25.00 and 52.93 ± 11.21 (P < 0.05) respectively at 60 s after TRAP treatment. The peaks of [Ca²(+)]i of D- and DV cells were 40.71 ± 5.89 and 61.07 ± 21.36 (P < 0.05) respectively at 60 s after thrombin treatment, but were 84.98 ± 11.23 and 102.58 ± 21.48 (P < 0.05) respectively at 40 s after TRAP treatment.</p><p><b>CONCLUSIONS</b>The high metastatic potential of PLA801D and PLA801C may be related to [Ca²(+)]i of the tumor cells. PAR-1 may play an important role in the metastasis of lung giant cell carcinoma cells by up-regulating the intracellular Ca²(+).</p>

Functional aspects of protease-activated receptor 1 in promoting metastasis of lung cancer / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the functional aspects of protease-activated receptor 1 (PAR-1) gene involved in tumor metastasis.</p><p><b>METHODS</b>Two human lung giant cell carcinoma cell lines PLA801C (low metastasis potential) and PLA801D (high metastasis potential) were chosen as in-vitro human cancer model systems. Sense and anti-sense expression constructs of PAR-1 gene (pC/PAR1s and pC/PAR1as) were transfected into PLA-801C and PLA-801D cells by lipofection. PAR-1 expression was determined by RT-PCR and western blot analysis. MTT growth, flow cytometry analysis, fibronectin adhesion, and matrigel invasion assays were used to study the effect of PAR-1 expression on the proliferation, adhesion, and invasion of the transfected cells.</p><p><b>RESULTS</b>Appropriate up-regulation or down-regulation of protein expression of PAR-1 was observed in both transfected cell lines (PLA801C and PLA801D) to express PAR-1s or PAR-1as, respectively. Expression of the sense PAR-1 markedly increased cellular proliferation, adhesion and invasion of PLA-801C cells. In contrast, anti-sense PAR-1 significantly inhibited cell growth, adhesion and invasion capabilities, along with cell arrest at G0/G1 phase of the PLA-801D cells.</p><p><b>CONCLUSIONS</b>Successful up- and down- regulation of expression of PAR-1 can be achieved by in-vitro transfection of sense and antisense PAR-1 constructs. PAR-1 may enhance metastasis of lung cancer through its regulation of cellular proliferation, adhesion and invasion. Down-regulation of expression of PAR-1 may provide a new therapeutic strategy against lung carcinoma.</p>

Expression of PAR-1 in human lung carcinoma and its relationship with tumor metastatic potential / 中华病理学杂志

<p><b>OBJECTIVE</b>To explore the correlation between expression of PAR-1 and metastasis of human lung carcinoma.</p><p><b>METHODS</b>Expression levels of PAR-1 were examined in surgically resected lung carcinoma specimens and corresponding lymph nodes by RT-PCR and immunohistochemistry, combined with morphometric methodology and clinicopathologic profiles.</p><p><b>RESULTS</b>Strong PAR-1 staining was detected in the periphery of carcinoma nests, adenocarcinomatous emboli, foci of atypical adenomatous hyperplasia adjacent to the adenocarcinoma and atypical proliferation of duct epithelium of bronchial mucous glands. The expression rates of PAR-1 were 73.8% (59/80) and 63.9% (23/36) by immunohistochemistry and RT-PCR respectively. The percentage of PAR-1 protein expression cells was significantly higher in tumors with metastasis (85.7%, 48/56) than those without (45.8%, 11/24). Morphometric study demonstrated that there were significant differences of PAR-1 protein expression levels between tumors with metastatic and those without, primary and metastatic carcinomas, primary carcinomas and benign lung tissues adjacent to the carcinoma. No significant correlation was found between PAR-1 expression level and tumor size, histological types and tumor grades. The positive rate of PAR-1 mRNA expression in the metastatic group was significantly higher than that of the non-metastatic group (78.3%, 18/23 v.s. 38.5%, 5/13).</p><p><b>CONCLUSION</b>PAR-1 expression may play an important role in determining the malignant phenotypes of lung cancers and significantly contribute to their initiation, progression and metastasis.</p>

Differentially expressed genes in human giant-cell lung cancer lines with different metastatic potentials / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To screen genes differentially expressed in two human giant-cell lung cancer lines of same origin but with different metastasis potentials.</p><p><b>METHODS</b>Suppression subtractive hybridization (SSH) was done twice on two giant-cell lung cancer lines, PLA-801C and PLA-801D (hereafter abbreviated as C and D), of same origin but with low (C) and high (D) metastatic potentials. In the first round, SSH C was used as tester and D as driver, while in the second round, the tester and driver were interchanged. The sequences acquired from both rounds of SSH were spotted on glass slides respectively and screened by hybridizing with two-color fluorescence probes. Clones that had different expression levels on chips were also confirmed by RNA dot blot or Northern blot.</p><p><b>RESULTS</b>There were 16 sequences with high expression in C as compared to those in D, and 79 sequences with high expression in D compared to those in C. After sequencing, most of them were found to be highly homologous to those encoding the following proteins: (1) cytokines and their receptors; (2) kinases and related proteins; (3) other proteins including enzymes, heat shock proteins, receptors, proteins of cell skeleton and mitochondria, products of oncogenes, etc; (4) some proteins deduced from gene sequences with yet unknown functions.</p><p><b>CONCLUSION</b>The alterations in expression of some known genes, including HSP70, AXL receptor tyrosine kinase and 14-3-3zeta, might have impact on metastasis of giant-cell lung cancer. Whether some differentially expressed genes newly revealed are metastasis-related needs further study.</p>

The specific killing of human melanoma cells by replication selective adenovirus / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To construct replication selective adenovirus AdhepE1 targeting human melanoma and observe its specific killing of human melanoma cells in vitro.</p><p><b>METHODS</b>Adenovirus E1 region, the murine tyrosinase promoter and enhancer DNA sequences were acquired respectively by PCR cloning. The shuttle plasmid of replication-selective adenovirus targeting human melanoma was constructed by DNA recombination. Replication-selective adenovirus AdhepE1 was generated by homologous recombination. The human melanoma cell line SK-Mel-1 and hepatocellular carcinoma cell line HepG2 were attacked separately by lower dose of AdhepE1. Change of cell morphology was observed and the surviving cells were calculated. The expression of E1A was assayed by RT-PCR to verify the specific-replication of AdhepE1.</p><p><b>RESULTS</b>Replication selective adenovirus AdhepE1 targeting human melanoma was acquired by PCR. Human melanoma cell line SK-Mel-1 was sensitive to oncolytic killing of AdhepE1 whereas HepG2 was little responsive. The results of RT-PCR suggested that AdhepE1 replicated specifically in human melanoma cells.</p><p><b>CONCLUSION</b>AdhepE1 can selectively kill human melanoma cells.</p>