Characterization of group Ⅰ metabotropic glutamate receptors in rat superior cervical ganglion and their changes following chronic intermittent hypoxia / 解剖学报

Objective To observe the expression and localization of group Ⅰ metabotropic glutamate receptors (mGluR1/ 5) in rat superior cervical ganglion (SCG) and the effect of chronic intermittent hypoxia (CIH) on mGluR1/ 5 protein level. Methods Twelve male SD rats were randomly divided into control group(Ctrl)and CIH group(CIH), 6 rats in each group. After 6 weeks of modeling, the effect of CIH on mGluR1/ 5 protein level was detected by Western blotting, the expression and distribution of mGluR1/ 5 in SCG were detected by immunohistochemistry and double-immunofluorescent staining. Results mGluR1/ 5 was expressed in rat SCG. mGluR1 was distributed in neurons and small intensely fluorescent (SIF) cells, but not in satellite glial cells (SGCs), nerve fibers and blood vessels, whereas mGluR5 was mainly distributed in nerve fibers and a little in neurons, but not in SGCs, SIF cells and blood vessels. CIH increased the protein levels of mGluR1/ 5 (P<0. 01) in rat SCG. Conclusion Both mGluR1 and mGluR5 are expressed in the rat SCG, but their distribution are different, and the increased protein levels of both may be involved in CIH-induced hypertension.

Cascading effects of seed-stem-individual spatial patterns along a grazing gradient.

Studying the seed trait-stem trait-individual spatial pattern system is helpful for understanding the developmental direction of plant dynamics and populations under grazing disturbance as well as the antagonistic relationship between animals and plants, but few systematic analyses of this spatial pattern system have been carried out. Kobresia humilis is the dominant species in alpine grasslands. We studied K. humilis seed traits and their relationship with K. humilis reproductive individuals, the relationship between reproductive and vegetative stems, and the weights and spatial patterns of reproductive and nonreproductive individuals under four grazing treatments: no grazing (control), light grazing, moderate grazing and heavy grazing. We explored the relationship among seed size and seed number with reproductive stems and vegetative stems along the grazing gradient and assessed the spatial pattern changes between reproductive and nonreproductive individuals. The results showed the following: (1) Seed size increased with increasing grazing intensity, and the coefficient of variation for seed size and seed number in the heavy grazing treatment was greater than 0.6. (2) The structural equation model showed that grazing treatment had a positive effect on seed number, seed size and reproductive stem number and a negative effect on reproductive stem weight. (3) Grazing treatment did not affect the resource allocation to reproductive stems and vegetative stems per unit length of reproductive K. humilis individuals. (4) Compared with the number of reproductive individuals in the no grazing treatment, the number in the heavy grazing treatment decreased significantly, and the negative correlation between reproductive individuals and nonreproductive individuals changed from a full-scale negative correlation to a small-scale negative correlation and a large-scale positive correlation. Our study showed that grazing could activate and change the resource allocation pattern of dominant species in a grassland and have significant positive effects on reproductive stem number, reproductive stem weight, seed number and seed size. Along a grazing intensity gradient, with the increase in distance between reproductive and nonreproductive individuals, the transformation of intraspecific relationships from a negative correlation to a positive correlation is an ecological strategy conducive to population survival.

Activation of metabotropic glutamate receptor 1 inhibits chronic intermittent hypoxia-induced carotid body plasticity in rats / 生理学报

The purpose of the present study was to explore the role of carotid body metabotropic glutamate receptor 1 (mGluR1) in chronic intermittent hypoxia (CIH)-induced carotid body plasticity. Sprague Dawley (SD) rats were exposed to CIH (6%-21% O2, 4 min/cycle, 8 h/day) for 4 weeks. The blood pressure of rats was monitored non-invasively by tail-cuff method under consciousness. RT-qPCR was used to examine the mRNA expression level of mGluR1 in rat carotid body. Western blot was used to detect the protein expression level of mGluR1 in rat carotid body. The role of mGluR1 in CIH-induced carotid body sensory long-term facilitation (sLTF) was investigated by ex vivo carotid sinus nerve discharge recording, and the carotid body sLTF was evoked by a 10-episode of repetitive acute intermittent hypoxia (AIH: 1 min of 5% O2 interspersed with 5 min of 95% O2). The results showed that: 1) CIH increased the systolic blood pressure (P < 0.001), diastolic blood pressure (P < 0.005) and mean arterial blood pressure (P < 0.001) of rats; 2) CIH decreased the mRNA and protein levels of mGluR1 in the rat carotid body (P < 0.01); 3) 4 weeks of CIH induced carotid body sLTF significantly, exhibiting as an increasing baseline sensory activity during post-AIH, which was inhibited by application of an agonist of group I metabotropic glutamate receptors, (S)-3,5-dihydroxyphenylglycine (DHPG), during sLTF induction (P < 0.005). In summary, these results suggest that activation of mGluR1 inhibits CIH-induced carotid body plasticity in rats.

Glutamate and its ionotropic receptor agonists inhibit the response to acute hypoxia in carotid body of rats / 生理学报

The purpose of this study was to investigate the effect of glutamate and its ionotropic receptor agonists on the response to acute hypoxia in rat carotid body in vitro. Briefly, after SD rats were anesthetized and decapitated, the bilateral carotid bifurcations were rapidly isolated. Then bifurcation was placed into a recording chamber perfused with 95% O2-5% CO2 saturated Kreb's solution. The carotid body-sinus nerve complex was dissected, and the carotid sinus nerve discharge was recorded using a suction electrode. To detect the response of carotid body to acute hypoxia, the chamber was perfused with 5% O2-5% CO2-90% N2 saturated Kreb's solution for a period of 100 s at an interval of 15 min. To observe the effect of glutamate, ionotropic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor agonist AMPA or N-methyl-D-aspartate (NMDA) receptor agonist NMDA on the response to acute hypoxia in rat carotid body, the chamber was perfused with 5% O2-5% CO2-90% N2 saturated Kreb's solution containing the corresponding reagent. The results showed that glutamate (20 μmol/L), AMPA (5 μmol/L) or NMDA (10 μmol/L) inhibited the acute hypoxia-induced enhancement of carotid sinus nerve activity, and these inhibitory effects were dose-dependent. In summary, the activation of glutamate ionotropic receptors appears to exert an inhibitory effect on the response to acute hypoxia in carotid body of rats.

Role of group II and III mGluRs in carotid body plasticity induced by chronic intermittent hypoxia in rats / 生理学报

The aim of the present study was to explore the role of group II and III metabotropic glutamate receptors (mGluRs) in carotid body plasticity induced by chronic intermittent hypoxia (CIH) in rats. Sprague Dawley (SD) rats were treated with CIH in Oxycycler A84 hypoxic chamber for 4 weeks, and the tail artery blood pressure was measured at the end of model preparation. RT-qPCR was performed to examine the mRNA expression levels of mGluR2/3/8 in rat carotid body. Carotid sinus nerve activity was detected by ex vivo carotid sinus nerve discharge recording technique, and acute intermittent hypoxia (AIH) was administered to induce carotid body sensory long-term facilitation (sLTF), in order to observe the role of group II and group III mGluRs in carotid body plasticity induced by CIH. The results showed that: 1) After 4 weeks of CIH exposure, the blood pressure of rats increased significantly; 2) CIH down-regulated the mRNA levels of mGluR2/3, and up-regulated the mRNA level of mGluR8 in the carotid body; 3) AIH induced sLTF in carotid body of CIH group. In the CIH group, activation of group II mGluRs had no effect on sLTF of carotid body, while activation of group III mGluRs completely inhibited sLTF. These results suggest that CIH increases blood pressure in rats, and group III mGluRs play an inhibitory role in CIH-induced carotid body plasticity in rats.

Improvement of high-quality evaluation criteria of Chinese patent medicines based on whole process control / 中国中药杂志

Chinese patent medicines(CPMs) are unique therapeutic drugs in China. Establishing and improving the evaluation criteria is an important measure to promote the high-quality development of CPMs. Based on the "evaluation criteria of high-grade CPMs with quality as the core index" established by our group in 2018, the "high-quality evaluation criteria for CPMs based on whole process control" was proposed in the present study in 2022. The scope of application and basic principles of the new criteria were clarified. A quality evaluation scoring table was established in the new criteria, including five parts: raw material selection, production process, quality control, efficacy evaluation, and brand building. The technical evaluation indexes involved have increased from 20% in the original criteria to 70% in the new criteria, and efficacy evaluation has been added in the new criteria. The subjective evaluation indicators account for a large proportion in the original criteria, which is prone to bias. The improved criteria overcome this shortcoming. It is expected that the new criteria as a basis can play a better role in the selection of high-quality products of CPMs, guide enterprises and institutions to participate actively in the evaluation and research of high-quality CPMs, and promote the high-quality development of CPMs.

Knockdown of ACC1 promotes migration of esophageal cancer cell / 中华肿瘤杂志

Objective: To investigate the effect of acetyl-CoA carboxylase 1 (ACC1) knockdown on the migration of esophageal squamous cell carcinoma (ESCC) KYSE-450 cell and underlying mechanism. Methods: Lentiviral transfection was conducted to establish sh-NC control cell and ACC1 knocking down cell (sh-ACC1). Human siRNA HSP27 and control were transfected by Lipo2000 to get si-HSP27 and si-NC. The selective acetyltransferase P300/CBP inhibitor C646 was used to inhibit histone acetylation and DMSO was used as vehicle control. Transwell assay was performed to detect cell migration. The expression of HSP27 mRNA was examined by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and the expressions of ACC1, H3K9ac, HSP27 and epithelial-mesenchymal transition-related proteins E-cadherin and Vimentin were detected by western blot. Results: The expression level of ACC1 in sh-NC group was higher than that in sh-ACC1 group (P<0.01). The number of cell migration in sh-NC group was (159.00±24.38), lower than (361.80±26.81) in sh-ACC1 group (P<0.01). The protein expression levels of E-cadherin and Vimentin in sh-NC group were statistically significant compared with sh-AAC1 group (P<0.05). The migrated cell number in sh-NC+ si-NC group was (189.20±16.02), lower than (371.60±38.40) in sh-ACC1+ si-NC group (P<0.01). The migrated cell number in sh-NC+ si-NC group was higher than that in sh-NC+ si-HSP27 group (152.40±24.30, P<0.01), and the migrated cell number in sh-ACC1+ si-NC group was higher than that in sh-ACC1+ si-HSP27 group (P<0.01). The protein expression levels of E-cadherin and Vimentin in sh-NC+ si-NC group were significantly different from those in sh-ACC1+ si-NC and sh-NC+ si-HSP27 groups (P<0.01). The protein expression levels of E-cadherin and Vimentin in sh-ACC1+ si-NC group were significantly different from those in sh-ACC1+ si-HSP27 group (P<0.01). After 24 h treatment with C646 at 20 μmmo/L, the migrated cell number in sh-NC+ DMSO group was (190.80±11.95), lower than (395.80±17.10) in sh-ACC1+ DMSO group (P<0.01). The migrated cell number in sh-NC+ DMSO group was lower than that in sh-NC+ C646 group (256.20±23.32, P<0.01). The migrated cell number in sh-ACC1+ DMSO group was higher than that in sh-ACC1+ C646 group (87.80±11.23, P<0.01). The protein expressions of H3K9ac, HSP27, E-cadherin and Vimentin in sh-NC+ DMSO group were significantly different from those in sh-ACC1+ DMSO group and sh-NC+ C646 group (P<0.01). The protein expression levels of H3K9ac, HSP27, E-cadherin and Vimentin in sh-ACC1+ DMSO group were significantly different from those in sh-ACC1+ C646 group (P<0.01). Conclusion: Knockdown of ACC1 promotes the migration of KYSE-450 cell by up-regulating HSP27 and increasing histone acetylation.

Effects of acetyl-CoA carboxylase 1 on proliferation, migration and invasion of glioma cell line U87 / 解剖学报

Objective To explore the effect of acetyl-CoA carboxylase 1(ACC1) on cell proliferation, migration and invasion of human glioma cell line U87. Methods Western blotting was performed to examine endogenous ACC1 expression in human glioma cell lines U87, U251 and U373. ACC1 overexpression plasmid and the plasmid vector were transiently transfected into U87 cells. The level of ACC1 in control and ACC1 overexpression cells was examined by Western blotting. The effect of ACC1 on U87 cells migration and invasion was detected by Transwell assay. The effect of ACC1 on U87 cells scratch healing ability was detected by scratch test. The effect of ACC1 on U87 cells proliferation was investigated by MTT assay. Western blotting was conducted to detect the level changes of proteins. Results Among three human glioma cell lines U87, U251 and U373, endogenous ACC1 level in U87 cells was lower than that in other two cell lines. ACC1 overexpression inhibited U87 cell proliferation, as well as cell migration, invasion and scratch healing ability (P < 0.05). Vimentin, fibronectin, urokinase type plasminogen activator (uPA), Bcl-2, cyclin B, cyclin D and p-STAT3 were down-regulated (P< 0.05), P21 was up-regulated (P < 0.05) after ACC1 overexpression. Conclusion These results suggest that ACC1 suppresses the proliferation, migration and invasion of human glioma cells, probably by inhibiting STAT3 activity.

Expression of β-site amyloid precursor protein cleaving enzyme 1 in rat superior cervical ganglion tissues / 解剖学报

Objective To observe the expression and localization of β-site amyloid precursor protein cleaving enzyme 1 (BACEl) in rat superior cervical ganglion and the effect of chronic intermittent frypoxia (CIH) on BACEl level. Methods The expression and distribution of BACEl in superior cervical ganglion were detected by RT-PCR, Western blotting and immunohistochemistry. Totally 16 male SD rats were randomly divided into control group and CIH group, 8 rats in each group. After 2 weeks of modeling, the effect of CIH on BACEl and peroxisome proliferators activated receptor gamma coactivator 1 alpha (PGC-la) mRNA level was detected by RT-PCR. Results BACEl was expressed in rat superior cervical ganglion, and mainly distributed in satellite glial cells and nerve fibers, but not in blood vessels, neurons and small intesely fluorecent(SIF) cells. CIH down-regulated BACEl mRNA level, but up-regulated PGC-la mRNA level ( P < 0.01). Conclusion BACEl is located in satellite glial cells and nerve fibers in the superior cervical ganglion of rats. The decreased level of BACEl ma)' be involved in the regulation of CIH-induced synaptic plasticity of superior cervical ganglion.

Research on the mechanism of hypoxia promoting the migration of lung adenocarcinoma A549 cells / 中国应用生理学杂志

Objective: To investigate the mechanism that hypoxia promotes the migration of lung adenocarcinoma A549 cells. Methods: A549 cells were cultured and cells that knockdown of acetyl-CoA carboxylase 1 (ACC1) were obtained by transfection with lentivirus, and cells that knockdown of sterol regulatory element-binding proteins-1 (SREBP-1) were obtained by treated with si-RNA. A549 cells were treated with hypoxia combined with hypoxia inducible factor-1α (HIF-1α) inhibitor PX-478 (25 μmol); Hypoxia combined with linoleic acid (LA) (20 μmol) treated A549 cells with ACC1 knockdown, and A549 cells with SREBP-1 knockdown were treated by hypoxia. Transwell migration assay was used to detect cell migration. Western blot was conducted to detect HIF-1α, ACC1 and epithelial mesenchymal transition (EMT) related proteins, Vimentin, E-Cadherin and SREBP-1; Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) was performed to detect the changes of ACC1 and SREBP-1 mRNA in A549 cells after hypoxia and HIF-1α inhibitor PX-478 (25 μmol) treatment. Each experiment was repeated three times. Results: Compared with the normoxic control group, hypoxia promoted the migration of A549 cells (P＜0.01), and up-regulated the expressions of ACC1, HIF-1α (all P＜0.01) and SREBP-1 (P＜0.05). PX-478 (25 μmol) inhibited the migration of A549 cells induced by hypoxia and down-regulated the expression of SREBP-1 (all P＜0.05). ACC1 mRNA and SREBP-1 mRNA levels were increased after hypoxia treatment of A549 cells (all P＜0.05). The levels of ACC1 mRNA and SREBP-1 mRNA were decreased after A549 cells treated with hypoxia combined with PX-478 (25 μmol) for 24 h (P＜0.05, P＜0.01). Knockdown of SREBP-1 in A549 cells was obtained by transfection with si-RNA. Transwell migration assay showed the number of cell migration in si-SREBP-1 group was less than that in normoxia control group (P＜0.01). The si-SREBP-1 group and the si-NC group were treated with hypoxia. Compared with the control group, the number of cell migration in the si-SREBP-1 group was decreased (P＜0.01), however, the difference was not statistically significant compared with the normoxia si-SREBP-1 group (P＞0.05). Western blot showed that the expression of ACC1 in the si-SREBP-1 group was lower than that in the control group (P＜0.01). Compared with the control group, the expression of ACC1 was decreased after si-SREBP-1 group treated with hypoxia (P＜0.01). Knockdown of ACC1 inhibited the migration of A549 cells (P＜0.05). After knockdown of ACC1, the migration number of A549 cells under normoxia and 5% O2 conditions had no significant difference (P＞0.05). Application of LA under hypoxia condition rescued ACC1-knockdown induced inhibitory effect on hypoxia-promoted A549 cell migration (P＜0.05). Conclusion: Hypoxia promotes migration of lung adenocarcinoma A549 cells by regulating fatty acid metabolism through HIF-1α/SREBP-1/ACC1 pathway.

Hypoxia promotes lung adenocarcinoma A549 cells migration by upregulating acetyl-CoA carboxylase 1 / 解剖学报

Objective To investigate the mechanism of hypoxia to promote human lung adenocarcinoma A549 cells migration through acetyl-CoA carboxylase 1 (ACCI). Methods Lung adenocarcinoma A549 cells were treated with hypoxia (5% 02 ). Transwell migration assay was used to detect cell migration ability. Western blotting was used to detect ACCI expression and epithelial-mesenchymal transition (EMT) related protein expression. Results Compared with the normoxia (control group), hypoxia treatment promoted the migration of A549 cells (P<0.01), ACCI expression was up- regulated after hypoxia treatment (P<0.01), and vimentin expression was detected to increase significantly (P<0.05), E- cadherin expression decreased (P<0.01) ; Compared with the control group, migration of A549 cells was inhibited (P<0.05), vimentin expression was down-regulated (P<0.05), and E-cadherin expression increased after knocking down ACC1(P<0.01). After ACCI was knocked down, the differences between the numbers of migration of A549 cells under normoxia and 5% 0

Trichostatin A promotes the migration of esophageal squamous carcinoma cells by protein kinase C signaling pathway / 解剖学报

Objective To investigate the effect of histone deacetylase inhibitor trichostatin A (TSA) on the migration of human esophageal squamous carcinoma cells(ESCC) and the possible mechanism. Methods KYSE-150 cells and EC9706 cells were cultured and Transwell assay was performed to detect the role of TSA alone and combined with protein kinase C (PKC) inhibitor AEB071 on cell migration; the images of morphology after cells treatment with TSA or combination of AEB071 with TSA; Western blotting was conducted to examine the protein level of epithelial-mesenchymal transition (EMT) related signaling molecules. Results TSA promoted the migration of ESCC cells significantly, and PKC inhibitor AEB071 partly inhibited the effect of TSA-promoted ESCC cells migration. Treatment with TSA resulted in the cell morphology transitioned from epithelia oval-like to mesenchymal spindle-like, indicating the EMT. AEB071 partially rescued ESCC cells morphological changes which TSA induced. Western blotting showed that TSA reduced the expression of E-cadherin and augmented the expression of vimentin, β-catenin, Slug and acH3, whereas AEB071 obviously blocked the EMT-related protein level changes which induced by TSA. Conclusion TSA promotes ESCC cells migration via inducing EMT process and the mechanism may be mediated by PKC signaling pathway.

Effect of cyclic intermittent hypoxia on the expression of β-site amyloid precursor protein cleaving enzyme 1 in rat adrenal gland / 解剖学报

Objective To observe the expression and localization of β-site amyloid precursor protein cleaving enzyme 1 (BACE1) in rat adrenal gland and to detect the effect of cyclic intermittent hypoxia (CIH) on the expression of BACE1. Methods The expression and localization of BACE1 in rat adrenal gland were detected by Western blotting and immunohistochemistry. Sixteen male Sprague-Dawley (SD) rats were randomly divided into two groups: control group and CIH group, 8 rats in each group. The protein levels of BACE1 and tyrosine hydroxylase (TH) in rat adrenal medulla were detected by Western blotting after CIH 2 weeks treatment. Results BACE1 was mainly localized in rat adrenal medullary nerve fibers. Compared with the control group, BACE1 protein level decreased and TH protein level increased in the adrenal medulla in the CIH group. Conclusion BACE1 is located in rat adrenal medullary nerve fibers. The decreased level of BACE1 may participate in slowing down the excessive enhancement of sympathetic activity induced by CIH.

Evaluation and analysis of high-quality products of Liuwei Dihuang Pills / 中国中药杂志

Liuwei Dihuang Pills is a typical traditional Chinese medicine with "the same product made by many manufacturers". The quality and price of products made in various factories was different obviously. In this study, the quality differences of Liuwei Dihuang Pills were evaluated over multi-dimensions and throughout the whole production cycle involving raw materials, production process, quality control and post-marketing re-studies based on the "Chinese patent medicine evaluation standard with quality at the core" established earlier by our research group. The research results showed that the product quality of various manufacturers was significantly different, and the product quality was positively correlated with the market shares of enterprises, indicating that enterprises with more market shares paid more attention to product quality; and the sales determined the concern degree of enterprises on products, which was in line with general cognition. During the single-item evaluation of Liuwei Dihuang Pills, the score of raw material selection was relatively low, and the enterprises paid less attention to the initial raw materials. The whole production process was better, and the national compulsory implementation of "Production Quality Management Standard"(GMP) had a positive effect in improving product quality. Quality control could basically guarantee product quality. Intelligent manufacturing promoted by the government was beneficial to ensure product uniformity. The score rate of "quality evaluation" item was basically qualified, which indicated that the quality control level of Liuwei Dihuang Pills was acceptable as a whole, but there was still room for improvement. "Re-evaluation and Brand Construction" had the lowest scores, reflecting that enterprises did not pay enough attention to in-depth study and re-evaluation of "the same product made by many manufacturers". The evaluation results were in line with expectations, provided a reference example for the evaluation of high-quality Chinese patent medicine, a basis for the high-quality and good price of Chinese patent medicine, scientific data for improving medical insurance bidding, and thus facilitated promoting the healthy development of the traditional Chinese medicine industry.

In Vitro Efficacy of Continuous Mild High Temperature on the Biofilm Formation of Aspergillus Niger / 中国医学科学院学报

Objective To investigate whether continuous mild high temperature (increased temperature without causing significant damage to host cells) can inhibit the biofilm formation of Aspergillus niger (A.niger) and its vitality.Methods A.niger biofilms were formed on a coverslip in 24-well tissue culture plate and were checked at the time points 4,8,10,16,24,48 and 72 hours.Confocal laser scanning microscopy (CLSM) was used to image and quantify A.niger biofilm formation under three different continuous mild high temperatures at 37℃,39℃,and 41℃.Furthermore,2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay was used to quantify the dynamic growth of A.niger biofilm under the above conditions.Results Compared with the culture condition 37℃,CLSM analysis at 39℃ or 41℃ showed that higher temperature induced later germination at 4 hours (t=8.603,P=0.047;t=14.550,P=0.008),poorer hyphal elongation at 8 hours(t=35.118,P=0.039;t=63.450,P=0.006),poorer polar growth,and reduced biofilm thickness from 10 to 24 hours.The XTT assay showed that higher temperature (39℃ or 41℃) lead to lower vitality at 10 hours,higher vitality at 16 hours,but finally lower vitality from 24 to 72 hours (t=24.262,P=0.038;t=7.556,P=0.031).Conclusion Continuous mild high temperature may have a negative regulatory effect on biofilm formation of A.niger and its vitality.

Establishment of a novel method for primary culture of normal human cervical keratinocytes / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Cervical keratinocytes are recovered at a low numbers and frequently associated with contaminating human fibroblasts which rapidly overgrow the epithelial cells in culture with medium supplemented with 10% fetal bovine serum (FBS). However, it is difficult to initiate keratinocyte cultures with serum-free keratinocyte growth medium alone because cell attachment can be poor. Therefore, the culture of these cells is extremely difficult. In this study, we described a modified culture medium and coated culture plastics for growing normal human cervical epithelial cells in vitro.</p><p><b>METHODS</b>Normal cervical epithelial tissue pieces were obtained and digested with type I collagenase to dissociate the cells and a single cell suspension produced. The cells were cultured on plastic tissue culture substrate alone or substrate coated with collagen type I from rat tail, with modified keratinocyte serum-free medium (K-SFM) supplemented with 5% FBS. After attachment, the medium were replaced with K-SFM without FBS. The expression of basal keratins of the ectocervical epithelium, K5, K14 and K19 were assayed by immunofluorescence with monoclonal antibodies to identify the cell purity.</p><p><b>RESULTS</b>Our results indicate that cells attached to the culture plastic more quickly in K-SFM supplemented with 5% FBS than in K-SFM alone, as well as to tissue culture plastic coated with collagen type I than plastic alone. The modified medium composed of K-SFM and 5% FBS combined with a specific tissue culture plastic coated with collagen type I from rat tail was the best method for culture of normal cervical epithelial cells. K5, K14 and K19 were assayed and keratinocyte purity was nearly 100%.</p><p><b>CONCLUSION</b>A novel, simple and effective method can be used to rapidly obtain highly purified keratinocytes from normal human cervical epithelium.</p>

The level of peripheral circular DNA in patients with hand foot and mouth disease / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To determine the circular DNA level of patients with hand foot and mouth disease (HFMD) and evaluate its potential clinical value.</p><p><b>METHODS</b>Venous blood in 30 healthy children and 78 patients with HFMD within 3 days of onset of illness and convalescent period was collected. The level of plasma circular DNA was detected by duplex real-time polymerase chain reaction assay. Blood sugar, high-sensitive CRP(hs-CRP) and leucocyte were also detected.</p><p><b>RESULTS</b>The level of circular DNA in control group was (6.57 +/- 4.67) ng/ml. The level of circular DNA in ordinary and severe HFMD patients was (11.51 +/- 7.75) ng/ml and (20.59 +/- 10.67) ng/ml before treatment, respectively. The levels of circular DNA in ordinary and severe HFMD patients were significantly higher than that in control group (P = 0.021; 0.000); the level of circular DNA in severe HFMD patients was significantly higher than that in ordinary HFMD patients (P = 0.011). The level of circular DNA in severe HFMD patients after treatment were significantly lower than that before treatment (P = 0.033). The level of circular DNA before treatment and after treatment in ordinary HFMD patients had no significant difference. The levels of blood sugar and hs-CRP in severe HFMD patients were higher than those in ordinary before treatment (P = 0.045; 0.011). The levels of blood sugar and hs-CRP before treatment and after treatment in ordinary HFMD patients had no significant change. There was significantly positive correlation between the level of circular DNA and that of hs-CRP in HFMD patient (P = 0.021), but there was no correlation between the level of circular DNA and that of blood sugar and leucocyte.</p><p><b>CONCLUSIONS</b>The level of circular DNA not only become an early identification marker of severe HFMD patients, but also become monitoring marker of effect of treatment.</p>