Why were doctors unable to accomplish their rural-aid mission in China? A qualitative study.

BACKGROUND: To alleviate the difficulties of rural residents in receiving timely healthcare, the Chinese government launched a medical rural-aid program that solicited urban medical professionals to go to rural hospitals for a 1-year tenure. However, many of urban doctors did not accomplish this task. In this study, we attempted to investigate the reasons behind the failure to fulfill this program and to explore a more feasible solution. METHODS: Eleven doctors and nurses participated in the focus group discussions. Twenty-five interviewees, including health administrative officials, doctors and managers from both urban tertiary hospitals and county-level hospitals, participated in semi-structured in-depth telephone interviews. The interview data were summarized and analyzed using the grounded theory. RESULTS: The failure of this program was attributed to multiple causes, such as problems with the recipient hospitals, the support hospitals and the participating doctors, and overall defects in the program strategy itself. One major reason is the competition between the recipient hospitals and the support hospitals, which distorted the original purpose of this rural-aid program. CONCLUSION: The rural-aid program strategy should be adjusted. The recipient hospitals should be township-level health centers rather than county-level hospitals. In addition, the relevant policies should be amended and improved accordingly. Copyright © 2015 John Wiley & Sons, Ltd.

Detection of IgG in sera of patients with schistosomiasis japonica by developing magnetic affinity enzyme-linked immunoassay based on recombinant 14-3-3 protein.

BACKGROUND: Low intensity of Schistosoma infection is the current status in China after long time treatment with praziquantel, therefore more sensitive diagnostic methods are required now. In this study, a magnetic affinity enzyme-linked immunoassay (MEIA) based on the signal transduction protein 14-3-3 of Schistosoma japonicum (Sj14-3-3), was developed for detecting schistosomiasis. METHODS: Sera of infected BALB/c mice were collected and analyzed with MEIA and ELISA. Both MEIA and ELISA based on Sj14-3-3 were further used to detect serum IgG in patients. Sera from 58 schistosomiasis-related patients with low-intensity infection, and 30 non-endemic negative controls, were collected to assess the assay. Six sera from paragonimiasis patients were used to analysis cross-reactions. RESULTS: Compared with ELISA, MEIA has a higher ratio of the mean positive value to the mean negative value (P/N) at the same dilution ratio in infected mice (3.71 vs 2.45). Similar results were observed in humans, higher P/N of MEIA compared to ELISA (3.57 vs 2.68). There was no cross-reaction with the sera of paragonimiasis patients detected by both MEIA and ELISA. CONCLUSIONS: Our studies suggested that MEIA based on recombinant Sj14-3-3 protein (rSj14-3-3) had the potential for the diagnosis of schistosomiasis.

An analysis of China's physician salary payment system.

Physician payment system (PPS) is a principal incentive system to motivate doctors to provide excellent care for patients. During the past decade, physician remuneration in China has not been in proportional to physician's average work load and massive responsibilities. This paper reviewed the constitution of the PPS in China, and further discussed the problems and issues to be addressed with respect to pay for performance. Our study indicated that the lower basic salary and bonus distribution tied to "profits" was the major contributor to the physician's profit-driven incentive and the potential cause for the speedy growth of health expenditures. We recommend that government funding to hospitals should be increased to fully cover physicians' basic salary, a flexible human resource and talent management mechanism needs to be established that severs personal interest between physicians and hospitals, and modern performance assessment and multiplexed payment systems should be piloted to encourage physicians to get the more legitimate compensation.

Tumor-derived IL-35 promotes tumor growth by enhancing myeloid cell accumulation and angiogenesis.

IL-35 is a member of the IL-12 family of cytokines that is comprised of an IL-12 p35 subunit and an IL-12 p40-related protein subunit, EBV-induced gene 3 (EBI3). IL-35 functions through IL-35R and has a potent immune-suppressive activity. Although IL-35 was demonstrated to be produced by regulatory T cells, gene-expression analysis revealed that it is likely to have a wider distribution, including expression in cancer cells. In this study, we demonstrated that IL-35 is produced in human cancer tissues, such as large B cell lymphoma, nasopharyngeal carcinoma, and melanoma. To determine the roles of tumor-derived IL-35 in tumorigenesis and tumor immunity, we generated IL-35-producing plasmacytoma J558 and B16 melanoma cells and observed that the expression of IL-35 in cancer cells does not affect their growth and survival in vitro, but it stimulates tumorigenesis in both immune-competent and Rag1/2-deficient mice. Tumor-derived IL-35 increases CD11b(+)Gr1(+) myeloid cell accumulation in the tumor microenvironment and, thereby, promotes tumor angiogenesis. In immune-competent mice, spontaneous CTL responses to tumors are diminished. IL-35 does not directly inhibit tumor Ag-specific CD8(+) T cell activation, differentiation, and effector functions. However, IL-35-treated cancer cells had increased expression of gp130 and reduced sensitivity to CTL destruction. Thus, our study indicates novel functions for IL-35 in promoting tumor growth via the enhancement of myeloid cell accumulation, tumor angiogenesis, and suppression of tumor immunity.

Magnetic affinity enzyme-linked immunoassay based on recombinant 26 kDa glutathione-S-transferase for serological diagnosis of schistosomiasis japonica.

Schistosomiasis remains a serious worldwide public health problem. Improving the diagnostic assay for surveillance and monitoring will contribute to hastening the possible elimination of the disease in endemic regions. Therefore, this study aims to develop magnetic affinity enzyme-linked immunoassay (MEIA) for serological diagnosis of schistosomiasis based on recombinant 26kDa glutathione-S-transferase of Schistosoma japonicum (rSj26GST). BALB/c mice infected with S. japonicum cercariae (40 per mouse) were used. After infecting for 6 weeks, the antibody was detected by MEIA. All of the infected mouse sera were effectively determined by MEIA. Compared with the enzyme-linked immunosorbent assay (ELISA), MEIA has a higher ratio of the mean positive value to the mean negative value (P/N) at the same dilution ratio (3.92 versus 2.66). MEIA was further applied for diagnosis of human schistosomiasis. Sera from 28 schistosomiasis-confirmed patients with low-intensity infection, 15 treated patients, and 20 non-endemic negative controls, were used to assess the assay. The results showed that MEIA and ELISA had similarity in positive detection rates. However, the higher P/N of MEIA was observed at the same dilution ratio. MEIA had high negative rate in detection of specific IgG in the treated patients. Moreover, there was no cross reaction with the sera of paragonimiasis patients. These results suggested that MEIA based on rSj26GST is a simple, rapid, convenient assay for the diagnosis of schistosomiasis.

Magnetic affinity enzyme-linked immunoassay for diagnosis of Schistosomiasis japonicum in persons with low-intensity infection.

Most schistosome-endemic areas in China are characterized by low-intensity infections that are independent of prevalence. To establish an effective diagnostic method, we developed a magnetic affinity enzyme-linked immunoassay based on soluble egg antigens (SEA-MEIA) for diagnosing schistosomiasis in persons with low-intensity infection with Schistosoma japonicum by comparing it with a conventional enzyme-linked immunosorbent assay (ELISA). Our results showed that the SEA-MEIA had a higher sensitivity and greater precision in the diagnosis of low-intensity S. japonicum infections than the ELISA. In addition, when we used Pearson's correlation in associating SEA-MEIA with ELISA, a significant correlation existed between the two assays (r = 0.845, P < 0.001). Our data indicated that SEA-MEIA, with a higher sensitivity and greater ease of performance, would be valuable for diagnosis of schistosomiasis japonicum in persons with low-intensity infections.

Cellular uptake and intracellular trafficking of PEG-b-PLA polymeric micelles.

Besides as an inert carrier for hydrophobic anticancer agents, polymeric micelles composed of di-block copolymer poly(ethylene glycol)-poly(lactic acid) (PEG-b-PLA) function as biological response modifiers including reversal of multidrug resistance in cancer. However, the uptake mechanisms and the subsequent intracellular trafficking remain to be elucidated. In this paper, we found that the uptake of PEG-b-PLA polymeric micelles incorporating nile red (M-NR) was significantly inhibited by both dynamin inhibitor dynasore and dynamin-2 dominant negative mutant (dynamin-2 K44A). Exogenously expressed caveolin-1 colocalized with M-NR and upregulated M-NR internalization in HepG2 cells expressing low level of endogenous caveolin-1, while caveolin-1 dominant negative mutant (caveolin-1 Y14F) significantly downregulated M-NR internalization in C6 cells expressing high level of endogenous caveolin-1. Exogenously expressed clathrin light chain A (clathrin LCa) did not mainly colocalize with the internalized M-NR and had no effect on M-NR uptake. These results suggested that dynamin- and caveolin-dependent but clathrin-independent endocytosis was involved in M-NR cellular uptake. We further found that M-NR colocalized with lysosome and microtubulin after internalization.

Endoplasmic reticulum Ca2+ release modulates endothelial nitric-oxide synthase via extracellular signal-regulated kinase (ERK) 1/2-mediated serine 635 phosphorylation.

Endothelial nitric-oxide synthase (eNOS) plays a central role in cardiovascular regulation. eNOS function is critically modulated by Ca(2+) and protein phosphorylation, but the interrelationship between intracellular Ca(2+) mobilization and eNOS phosphorylation is poorly understood. Here we show that endoplasmic reticulum (ER) Ca(2+) release activates eNOS by selectively promoting its Ser-635/633 (bovine/human) phosphorylation. With bovine endothelial cells, thapsigargin-induced ER Ca(2+) release caused a dose-dependent increase in eNOS Ser-635 phosphorylation, leading to elevated NO production. ER Ca(2+) release also promoted eNOS Ser-633 phosphorylation in mouse vessels in vivo. This effect was independent of extracellular Ca(2+) and selective to Ser-635 because the phosphorylation status of other eNOS sites, including Ser-1179 or Thr-497, was unaffected in thapsigargin-treated cells. Blocking ERK1/2 abolished ER Ca(2+) release-induced eNOS Ser-635 phosphorylation, whereas inhibiting protein kinase A or Ca(2+)/calmodulin-dependent protein kinase II had no effect. Protein phosphorylation assay confirmed that ERK1/2 directly phosphorylated the eNOS Ser-635 residue in vitro. Further studies demonstrated that ER Ca(2+) release-induced ERK1/2 activation mediated the enhancing action of purine or bradykinin receptor stimulation on eNOS Ser-635/633 phosphorylation in bovine/human endothelial cells. Mutating the Ser-635 to nonphosphorylatable alanine prevented ATP from activating eNOS in cells. Taken together, these studies reveal that ER Ca(2+) release enhances eNOS Ser-635 phosphorylation and function via ERK1/2 activation. Because ER Ca(2+) is commonly mobilized by agonists or physicochemical stimuli, the identified ER Ca(2+)-ERK1/2-eNOS Ser-635 phosphorylation pathway may have a broad role in the regulation of endothelial function.

Delivery of TFPI-2 using ultrasound with a microbubble agent (SonoVue) inhibits intimal hyperplasia after balloon injury in a rabbit carotid artery model.

Here we report a new, simple and efficient method by using ultrasound and a microbubble agent (SonoVue) for delivering a gene to balloon-injured carotid arteries for restenosis prophylaxis. The tissue factor pathway inhibitor-2 (TFPI-2) has been shown to inhibit the postinjury intimae hyperplasia in atherosclerotic vessels. New Zealand white rabbits were divided into 4 groups with 14 in each, a treatment control for balloon injury, a gene vehicle control, a gene delivery of TFPI-2 without using ultrasound and a gene delivery of TFPI-2 using ultrasound. After four weeks, the injured artery neointimal proliferation was significantly lower in the TFPI-2 group with ultrasound than the control groups (p < 0.01) according to the measurement of the mean luminal diameters by B-mode ultrasonography. The ratio of intimal/media area and the stenosis rate in the gene delivery facilitated by ultrasound were significantly lower than those of the nonultrasound gene delivering method (p < 0.01).

Puerarin attenuates glutamate-induced neurofilament axonal transport impairment.

AIM OF THE STUDY: Puerarin (Pur) is a primary component of the most functional extracts of Pueraria lobata used in traditional Chinese medicine for centuries. Since it has been postulated that Pur protects the brain against glutamate (Glu) neurotoxicity, we investigated the effects of Pur on Glu-induced axonal transport impairment in primary hippocampal neurons in this study. MATERIALS AND METHODS: Primary hippocampal cultures were prepared from 2-day-old Sprague-Dawley rats. Intracellular calcium concentration [Ca(2+)](i), neurofilament (NF) phosphorylation and protein kinase activity for Cdk5 were measured. Time-lapse imaging technology was used to capture the NF axonal transport in the cultured neurons with transiently transfected fluorescence protein linked to the N-terminus of NF-M (EGFP-NFM). RESULTS: The results showed that Pur significantly diminished the Glu-induced elevation of [Ca(2+)](i) in dose-dependent manner and antagonized the Glu-evoked increases in NF phosphorylation at protein levels. The neurons under the Glu treatment displayed the accumulation of immobile NF clusters in the cell body and the reduced rates of axonal transport of NFs by 72.8% compared to the control neurons. Intriguingly, Pur reversed the slowed rate of the axonal transport by 35.6%. Pur also remarkably attenuated Glu-evoked activation of Cdk5. CONCLUSIONS: Pur may play a role in protecting against Glu-induced NF axonal transport impairment in rat primary hippocampal neurons by inhibiting the increased [Ca(2+)](i) and by impeding the activation of Cdk5.

Increased expression of cdk5/p25 in N2a cells leads to hyperphosphorylation and impaired axonal transport of neurofilament proteins.

AIMS: Alzheimer's disease (AD) is the leading cause of dementia. The increased cdk5 expression and enhanced phosphorylation of tau and NFs have been seen in AD patients. Our study aimed at investigating the effects of increased cdk5 activity on axonal transport of neurofilaments (NFs). MAIN METHODS: In this study, we used a molecular engineering approach to overexpress cdk5/p25 in neuroblastoma N2a cells and investigated the effects on axonal transport with live cell imaging techniques. KEY FINDINGS: In stably transfected cells, there was a 2.5-fold increase in cdk5 activity compared to non-transfected cells, which in turn led to a dramatic increase in phosphorylation of NFs and tau at several phosphorylation sites. Using time-lapse imaging technology, the transport of NFs was captured in the cells overexpressing cdk5/p25, which were also transiently transfected with fluorescence protein linked to the N-terminus of NF-M (EGFP-NFM). The cdk5/p25 cells displayed significantly slower rates of axonal transport of NFs, with accumulation of immobile NF clusters observed in the cell body. Roscovitine, an inhibitor of cdk5, significantly reversed this defect in axonal transport. SIGNIFICANCE: These results suggest that increased cdk5 activity found in AD subjects may be crucially related to the pathogenesis of AD via an underlying mechanism by which it promotes accumulation of excessively phosphorylated cytoskeletal NF proteins, leading to the enduring impairment of axonal transport of NFs.

Up-regulation of VLDL receptor expression and its signaling pathway induced by VLDL and beta-VLDL.

Very low density lipoprotein receptor (VLDLR) is thought to participate in the pathogenesis of atherosclerosis induced by VLDL and beta-VLDL. The present study was undertaken to elucidate the effects of VLDL and beta-VLDL on VLDLR expression and its signaling pathway. RAW264.7 cells were incubated with VLDL and beta-VLDL. The expression of VLDLR mRNA was detected by RT-PCR. The transcriptional activity of VLDLR gene was detected in recombinant plasmid pGL4.2VR-luciferase transfected RAW264.7. Western blot assay was used to detect the changes of phosphorylated ERK1/2 protein. Inhibitors or activators were used to observe the signal pathway involving VLDLR expression regulation. The results showed that VLDL and beta-VLDL stimulated ERK1/2 activity in a PKC-dependent manner. VLDL or beta-VLDL-induced VLDLR expression on macrophages was extremely abolished by inhibitors ERK1/2 or PKC. Our findings revealed that VLDL or beta-VLDL-induced VLDLR expression via PKC/ERK cascades and the effect was linked to the transcriptional activation of VLDLR gene promoter.

Up-regulation of VLDL Receptor Expression and Its Signaling Pathway Induced by VLDL and β-VLDL / 华中科技大学学报（医学）（英德文版）

Very low density lipoprotein receptor (VLDLR) is thought to participate in the patho-genesis of atherosclerosis induced by VLDL and β-VLDL. The present study was undertaken to elu-cidate the effects of VLDL and β-VLDL on VLDLR expression and its signaling pathway. RAW264.7 cells were incubated with VLDL and β-VLDL. The expression of VLDLR mRNA was detected by RT-PCR. The transcriptional activity of VLDLR gene was detected in recombinant plasmid pGL4.2VR-luciferase transfected RAW264.7. Western blot assay was used to detect the changes of phosphorylated ERK1/2 protein. Inhibitors or activators were used to observe the signal pathway in-volving VLDLR expression regulation. The results showed that VLDL and β-VLDL stimulated ERKI/2 activity in a PKC-dependent manner. VLDL or β-VLDL-induced VLDLR expression on macrophages was extremely abolished by inhibitors ERKI/2 or PKC. Our findings revealed that VLDL or β-VLDL-induced VLDLR expression via PKC/ERK cascades and the effect was linked to the transcriptional activation of VLDLR gene promoter.

Stem cell factor/c-kit signaling mediated cardiac stem cell migration via activation of p38 MAPK.

OBJECTIVE: It was reported that there are cardiac stem cells (CSCs) in the rat heart, and they could reconstitute well-differentiated myocardium that are formed by blood-carrying new vessels and myocytes. However, how do the CSCs migrate into the peri-infarcted areas after myocardial infarction (MI)? It remains entirely unknown about the signal transduction involved in the migration of CSCs. METHODS AND RESULTS: Rat heart MI was induced by left coronary artery ligation. Both immunohistochemical staining and Western blotting analysis was performed to detect the expression of SCF protein, and RT-PCR was conducted for the expression of SCF mRNA. Cardiac stem cells were isolated from rat hearts, and a cardiac stem cell migration assay was performed using a 48-well chemotaxis chamber system. On day 5 after MI in rats, the expression of stem cell factor (SCF) mRNA and protein was significantly increased in the peri-infarcted area, which was matched with more accumulation of CSCs in the region and improvement of cardiac function, which was blocked by p38 MAPK selective inhibitor SB203580. In in vitro experiments, SCF induced CSC migration in a concentration-dependent manner, and the antibody against SCF receptor (c-kit) blocked the SCF-induced CSC migration. Western blot analysis showed that the phosphorylated p38 MAPK (Phospho-p38 MAPK) was highly increased in the SCF-treated CSCs, and the inhibition of p38 MAPK activity significantly attenuated SCF-induced the migration of CSCs. CONCLUSION: It demonstrated that SCF/c-kit signaling may mediate the migration of CSCs via activation of p38 MAPK.

Effect of truncated-ApoE4 overexpression on tau phosphorylation in cultured N2a cells.

The carboxyl-terminal amino acids 272-299-truncated apoE4 (delta272-299) is the main fragments of apoE4 hydrolysate in neurons. The effects of truncated-ApoE4 (delta272-299) overexpression on tau phosphorylation in cultured N2a cells were investigated. The truncated-apoE4 (delta272-299) cDNA was subcloned into pEGFP-c3 to form recombinant pEGFP-T-apoE4. pEGFP-c3, pEGFP-T-apoE4 and pEGFP-apoE4 were transfected into N2a cells respectively by lipofectamine 2000 method. After 24--48 h, tau phosphorylation was detected by Western blot assay and glycogen synthase kinase-3 (GSK-3) activity by using GSK-3 activity assay. The results showed that the overexpression of both full length-apoE4 and truncated apoE4 fragments in N2a cells induced a dramatic increase in phosphorylation of tau at Ser202 sites and the activation of GSK-3 as compared with untransfected cells, most significantly in the cells transfected with pEGFP-T-apoE4 (P < 0.05). It was concluded that in vitro overexpression of truncated-ApoE4 (delta272-299) can result in tau hyperphosphorylation in N2a cells by activating GSK-3, suggesting truncated-ApoE4 (delta272-299) might contribute the pathogenesis of Alzheimer disease.

Overexpressed heat shock protein 70 protects cells against DNA damage caused by ultraviolet C in a dose-dependent manner.

Heat shock protein 70 (Hsp70) comprises proteins that have been reported to protect cells, tissues, and organisms against damage from a wide variety of stressful stimuli; however, little is known about whether Hsp70 protects against DNA damage. In this study, we investigated the relationship between Hsp70 expression and the levels of ultraviolet C (UVC)-induced DNA damage in A549 cells with normal, inhibited, and overexpressed Hsp70 levels. Hsp70 expression was inhibited by treatment with quercetin or overexpressed by transfection of plasmids harboring the hsp70 gene. The level of DNA damage was assessed by the comet assay. The results showed that the levels of DNA damage (shown as the percentage of comet cells) in A549 cells increased in all cells after exposure to an incident dose of 0, 10, 20, 40, and 80 J/m2 whether Hsp70 was inhibited or overexpressed. This response was dose dependent: a protection against UVC-induced DNA damage in cells with overexpressed Hsp70 was observed at UVC dose 20 J/m2 with a maximum at 40 J/m2 when compared with cells with normal Hsp70 levels and in quercetin-treated cells. This differential protection disappeared at 80 J/m2. These results suggest that overexpressed Hsp70 might play a role in protecting A549 cells from DNA damage caused by UVC irradiation, with a threshold of protection from at UVC irradiation-induced DNA damage by Hsp70. The detailed mechanism how Hsp70 is involved in DNA damage and possible DNA repair warrants further investigation.

Effect of GSK-3 overactivation on neurofilament phosphorylation.

In this study, we studied the effect of glycogen synthase kinase-3 (GSK-3) overactivation on neurofilament phosphorylation in cultured cells. After N2a cells were treated with the specific inhibitor (wortmannin) of phosphoinositol-3 kinase (PI-3K) or treated with wortmannin and the specific inhibitor (LiCl) of glycogen synthase kinase-3 (GSK-3), GSK-3 activity and neurofilament phosphorylation were detected by using GSK-3 activity assay, Western blots and immunofluoresence. Our results showed that after treatment of N2a cells with wortmannin for 1 h, overactivation of GSK-3 caused a reduced staining with antibody SMI32 and an enhanced staining with antibody SMI31. When N2a cells were treated with wortmannin and LiCl, the activity of GSK-3 was reduced substantially. At the same time, the phosphorylation of neurofilament was also reduced. The study demonstrated that overactivation of GSK-3 induced hyperphosphorylation of neurofilament and suggested that in vitro overactivation of GSK-3 resulted in neurofilament hyperphosphorylation and this may be the underlying mechanism for Alzheimer's disease.

Beta-VLDL induced VLDL-R's up-regulation via PKC-ERK1/2 signal pathway.

To explore the intracellular signal pathways for beta-VLDL induced very low density lipoprotein receptor (VLDL-R) transcription up-regulation and their effects on lipid accumulation in macrophages, Western Blot was used to examine phosphorylated ERK1/2 protein and regulated effects by different singal kinase inhibitants. It was found that beta-VLDL induced an increase in ERK1/2 activity in a protein kinase C (PKC)-dependent manner in murine RAW264.7 macrophages. By using different protein kinases inhibitors or activators, it was observed that the effect of beta-VLDL induced VLDL receptor transcription, which was monitored by RT-PCR analysis of VLDL receptor mRNA, was not affected by the inhibitor of p38 kinase and cAMP analog, but extremely abolished by pretreating cells with PD98059, an inhibitor of ERK and GF 109203X, an inhibitor of PKC. These results demonstrated that the PKC-ERK1/2 cascade is the essential signaling pathway by which beta-VLDL activated VLDL-R mRNA expression. Inhibition of the ERK1/2 signaling cascade resulted in suppression of the cellular lipid accumulation induced by beta-VLDL in macrophages.

The inhibitory effects of an antisense u-PAR vector on invasion of highly invasive human prostate carcinoma PC-3M cell subclones.

To observe the inhibitory effects of an antisense u-PAR vector on invasion of highly invasive PC-3M cell subclones, the effects of the antisense u-PAR on activity of MMP-9 in those highly invasive cell subclones were detected by a quantitative RT-PCR and zymography. The monolayer invasion assay and colony formation assay in soft agar were used. And tumorigenesis rate and invasions by the cell subclones with or without the antisense u-PAR were observed in nude mice. It was found that in vitro growth of highly invasive PC-3M cell subclones transfected with the antisense u-PAR was declined, and the ability of anchorage-independent growth of those cell subclones was found decreased sharply, with the inhibiting rate becoming 79% and 60%, respectively. Although the antisense u-PAR didn't change MMP-9 gene transcription, they could inhibit the activation of MMP-9 of highly invasive PC-3M cell subclones. Moreover, the tumorigenesis rate of the cell subclones with the antisense u-PAR decreased and the growth of a neoplasm also slowed down. The t tests showed the difference between experimental and control groups was statistically significant (P < 0.01). The antisense u-PAR vector could not only inhibit the invasion ability of highly invasive PC-3M cell subclones in vitro but also restrain the growth of those cell subclones in vivo.

Construction of antisense RNA expression plasmid for u-PAR and its transfection to highly invasive PC-3M cell subclones.

To evaluate the specific inhibition of antisense u-PAR on the u-PAR expressions in highly invasive cell subclones and to determine its blocking function in the invasion by those cells, a cDNA fragment of u-PAR obtained by RT-PCR was inserted into a plasmid vector named pcDNA3 in antisense orientation. Then the antisense u-PAR recombinant was transfected into highly invasive cell subclones. The u-PAR expression in neo-resistant cells was examined by RT-PCR and immunohistochemical assay. Compared to the control cells, the content of mRNA and protein of u-PAR in transfected cells decreased sharply, and the rate of inhibition was 53% and 73%, respectively, indicating that an antisense u-PAR might have played a specific inhibitory role in its expression in the cells, which may provide a good cell model for making further investigation of the inhibitory effects of the antisense u-PAR on invasion in highly invasive cell subclones of human prostate carcinoma.