Influence of Angiotensin II on α1-Adrenergic Receptors Function in Rat Aorta and Expression in Vascular Smooth Muscle Cells

Angiotensin II (Ang II) is the main mediator of the Renin-Angiotensin-System acting on AT1 and other AT receptors. It is regarded as a pleiotropic agent that induces many actions, including functioning as a growth factor, and as a contractile hormone, among others. The aim of this work was to examine the impact of Ang II on the expression and function of α1-adrenergic receptors (α1-ARs) in cultured rat aorta, and aorta-derived smooth muscle cells. Isolated Wistar rat aorta was incubated for 24 h in DMEM at 37˚C, then subjected to isometric tension and to the action of added norepinephrine, in concentration-response curves. Ang II was added (1 × 10−5 M), and in some experiments, 5-Methylurapidil (α1A-AR antagonist), AH11110A (α1B-AR antagonist), or BMY-7378 (α1D-AR antagonist), were used to identify the α1-AR involved in the response. Desensitization of the contractile response to norepinephrine was observed due to incubation time, and by the Ang II action. α1D-AR was protected from desensitization by BMY-7378;while RS-100329 and prazosin partially mitigated desensitization. In another set of experiments, isolated aorta-derived smooth muscle cells were exposed to Ang II and α1-ARs proteins were evaluated. α1D-AR increased at 30 and 60 min post Ang II exposure, the α1A-AR diminished from 1 to 4 h, while α1B-AR remained unchanged over 24 h of Ang II exposure. Ang II induced an increase of α1D-AR at short times, and BMY-7378 protected α1D-AR from desensitization.

Comparative Analysis of Ki-67 Protein as a Proliferative Expression Index in Cutaneous Basal and Squamous Cell Carcinoma in Federal Medical Centre Umuahia, Nigeria

Background: Evaluating the tumor proliferative index helps predict clinical behavior and provides prognostic insights for cutaneous basal cell carcinoma (cBCC) and squamous cell carcinoma (cSCC). Objective: This study aimed to identify differences in the proliferative indices among variants of cBCC and cSCC diagnosed at a tertiary healthcare center. Method: Skin biopsies histologically diagnosed as cBCC and cSCC between 2012 and 2018 at the Federal Medical Centre (FMC) Umuahia, Abia State, Nigeria, were analyzed. Archival formalin-fixed, paraffin-embedded (FFPE) tissue blocks were retrieved along with clinical data, and were prepared on charged microscope slides and the immunohistochemical staining was carried out. The primary antibody used in this study was clone BioCare CRM325C (RM) and adenotonsillar tissue blocks/slides served as positive controls. Ki-67 immunohistochemistry was performed on fresh 4µm sections of the tumor specimens. Results: The application of Ki-67 immunoperoxidase on both BCC and SCC cohort, yielded an intense observable brownish nuclear stain in areas of dense proliferating tumour cells on both cutaneous tumours. The average Ki-67 index for all cSCC cases was 24.7%, with a range of 2.3% - 80%, while the mean for cBCC was 15.8%, ranging from 1.2% - 45.6%. Variants with high proliferative indices were observed in 11.9% of cBCC cases and 29.1% of cSCC cases. Among the low proliferative index category, cSCC accounted for 5.4%, while cBCC represented 14.3%. For mild proliferative indices, cSCC cases made up 7.3% and cBCC, 11.9%. The majority of cases showed moderate proliferative indices, with 61.9% for cBCC and 58.2% for cSCC. Overall, there was a significant difference in proliferative indices between cSCC, cBCC, and their variants. Conclusion: The study found a significantly higher rate of cell proliferation, measured by Ki-67 immunostaining, in cSCC and its variants compared to cBCC. However, certain variants of cBCC also exhibited high Ki-67 expression, indicating they can be as aggressive as some cSCC variants.

Sequential expression of miR-221-3p and miR-338-3p in Schwann cells as a therapeutic strategy to promote nerve regeneration and functional recovery

The functional properties of endogenous Schwann cells(SCs)during nerve repair are dynamic.Optimizing the functional properties of SCs at different stages of nerve repair may have therapeutic benefit in improving the repair of damaged nerves.Previous studies showed that miR-221-3p promotes the proliferation and migration of SCs,and miR-338-3p promotes the myelination of SCs.In this study,we established rat models of sciatic nerve injury by bridging the transected sciatic nerve with a silicone tube.We injected a miR-221 lentiviral vector system together with a doxycycline-inducible Tet-On miR-338 lentiviral vector system into the cavity of nerve conduits of nerve stumps to sequentially regulate the biological function of endogenous SCs at different stages of nerve regeneration.We found that the biological function of SCs was sequentially regulated,the diameter and density of myelinated axons were increased,the expression levels of NF200 and myelin basic protein were increased,and the function of injured peripheral nerve was improved using this system.miRNA Target Prediction Database prediction,Nanopore whole transcriptome sequencing,quantitative PCR,and dual luciferase reporter gene assay results predicted and verified Cdkn1b and Nrp1 as target genes of miR-221-3p and miR-338-3p,respectively,and their regulatory effects on SCs were confirmed in vitro.In conclusion,here we established a new method to enhance nerve regeneration through sequential regulation of biological functions of endogenous SCs,which establishes a new concept and model for the treatment of peripheral nerve injury.The findings from this study will provide direct guiding significance for clinical treatment of sciatic nerve injury.

Construction of retroviral vectors to induce a strong expression of human class Ⅰ interferon gene in human hepatocellular carcinoma cells in vitro

AIM To establish the hepatoma cell specific expression of human interferon gene mediated by retroviral vectors. METHODS Human interferon α and interforon β complementary DNA (IFNs cDNA) were cloned into polylinker site of pMNSM retroviral vector to construct recombinant retroviral vector pMNSIFNA and pMNSIFNB, where the transcription of IFN gene was driven by SV40 early region promoter, and pMNAIFNA, pAMNSIFNA and pMNAIFNB, where the transcription of IFN gene was driven by SV40 early region promoter regulated by α fetoprotein enhancer. The retroviral constructs were respectively introduced into retroviral amphotropic packaging cells by means of lipofectamine mediated gene transfer procedure. The plasmids transfection rate was (4～40)×10 3 colonies/μg DNA/10 6 PA317 cells. The retrovirus infection rate was (5～500)×10 4 colony forming units (CFU)/ml. The recombinant retroviruses were used to infect human hepatoma cells, renal carcinoma cells and melanoma cell lines in the presence of 4mg/L polybrene. RESULTS Northern and Dot hybridization of total RNA from the neomycin resistant colonies and interferon expression assay indicated that human α fetoprotein enhancer induced efficient and apecific transcription and expression of IFNs gene driven by the promoter of different origin in human hepatoma cells by which α fetoprotein was highly produced. CONCLUSION Cis active element of α fetoprotein gene can drive specific expression of IFNs gene in human hepatoma cells, which provides some valuable data for the hepatoma specific immune gene therapy.

The Effects of Triptolide on HLA Antigens Expression of Corneal Epithelial Cells Induced by Interferon-γ in Vitro

Objective:To observe the effects of immunosuppressants triptolide(TL)and cyclosporineA(CSA)on HLA antigens expression induced by interferon-γ（INF-γ）in vitro.Method:By using an indirect immunofluorescent method and analysing with ACAS-570,the abnormal HLA antigen expression of cultured corneal epithelial cells was induced by INF-γ，After incubation with one of the immunosuppressants(CSA,TL) for72hrs.the amount of HLA-ABCand HLA-DRantigens was measured.Result:There was no significant difference(P>0.05)between the group with CAS and the positive control group without CAS.In contrast to CSA,TL dramatically inhibited INF-γinduced expression of HLA antigens of corneal epithelial cells(P<0.001),compared with the control group withoutTL.Conclusion:TL had direct inhibition on the expression of HLA-ABCand HLA-DRantige ns induced by INF-γin vitro,while CSAhad no obvious inhibition,Eye Science2000;16:34-37.

Expression of Human Interferon α 2b Gene in Ginseng Cells

The human interferon α 2b（hIFN-α2b） gene was cloned into binary vector pBI121 to obtain plant expression vector pBIFN. The recombinant plasmid pBIFN was transferred into Agrobacterium tumefaciens strain LBA4404. Then the hIFN-α2b gene was introduced into ginseng callus cells via Agrobacterium-mediated transformation and the ginseng cell line carrying hIFN-α2b gene was selected on G418-containing medium. The presence of target gene in transformed cells was confirmed by PCR and RT-PCR. The results indicate that hIFN-α2b gene has been integrated into the ginseng cells＇ genome, with transcription products, hIFN-α2b expressed by the transgenic ginseng cells was detected by Western blot. It was shown that a specific protein band at 19000 could be observed. Cytopathic effect（CPE） inhibition assay using the W1SH-VSV system shows that the mean antiviral activity of expressed hlFN-a2α was 6.0× 10^4 IU/mL.

Astrocytic endothelin-1 overexpression impairs learning and memory ability in ischemic stroke via altered hippocampal neurogenesis and lipid metabolism

Vascular etiology is the second most prevalent cause of cognitive impairment globally.Endothelin-1,which is produced and secreted by endothelial cells and astrocytes,is implicated in the pathogenesis of stroke.However,the way in which changes in astrocytic endothelin-1 lead to poststroke cognitive deficits following transient middle cerebral artery occlusion is not well understood.Here,using mice in which astrocytic endothelin-1 was overexpressed,we found that the selective overexpression of endothelin-1 by astrocytic cells led to ischemic stroke-related dementia(1 hour of ischemia;7 days,28 days,or 3 months of reperfusion).We also revealed that astrocytic endothelin-1 overexpression contributed to the role of neural stem cell proliferation but impaired neurogenesis in the dentate gyrus of the hippocampus after middle cerebral artery occlusion.Comprehensive proteome profiles and western blot analysis confirmed that levels of glial fibrillary acidic protein and peroxiredoxin 6,which were differentially expressed in the brain,were significantly increased in mice with astrocytic endothelin-1 overexpression in comparison with wild-type mice 28 days after ischemic stroke.Moreover,the levels of the enriched differentially expressed proteins were closely related to lipid metabolism,as indicated by Kyoto Encyclopedia of Genes and Genomes pathway analysis.Liquid chromatography-mass spectrometry nontargeted metabolite profiling of brain tissues showed that astrocytic endothelin-1 overexpression altered lipid metabolism products such as glycerol phosphatidylcholine,sphingomyelin,and phosphatidic acid.Overall,this study demonstrates that astrocytic endothelin-1 overexpression can impair hippocampal neurogenesis and that it is correlated with lipid metabolism in poststroke cognitive dysfunction.

Molecular Cloning and Expression of RSSG58 Gene in Rice Sperm Cells

Myosins, a large family of structurally diverse mechanoenzymes, which, upon interaction with actin filaments, convert energy from ATP hydrolysis into mechanical force, play an important role in male reproductive processes. In this study we report the rice ( Oryza sativa L.) RSSG58 gene, which was cloned from the cDNA library of rice sperm cells by using sperm cell mainly expression subtractive clone as probe. This gene encodes a putative 66.7 W polypeptide, which shows similarity to the myosin heavy chain of Arabidopsis thaliana, and consists of 579 amino acids with an isoelectric point (pI) of 4.885. RSSG58, which is a member of a divergent gene family, generates transcripts of 2 278 bp and 2 437 bp that differ only in their polyadenylation sites. Southern hybridization showed that RSSG58 has only one copy in rice genome and RSSG58 transcripts are most abundant in sperm cells, with two distinct signals. The RT-PCR analysis indicated that the transcriptions of the RSSG58 gene were various in the different development stages and tissues. The greatest accumulation of RSSG58 mRNA was detected in sperm cells, while weaker expression was detected in leaves, microspore mother cells, unicellular microspore pollen stage, two-cell stage pollens, mature pollens and pollinated ovaries. These results suggest that RSSG58 is especially abundantly expressed in rice sperm cells.

Interferon-γ Alters the Immune-related miRNA Expression of Microvesicles Derived from Mesenchymal Stem Cells

Increasing studies have demonstrated that interferon gamma（IFN-γ）,which serves as a critical inflammatory cytokine,is essential to induce the immunosuppressive effects of mesenchymal stem cells（MSCs）.However,the mechanisms underlying the enhanced immunosuppressive effects of IFN-γ-stimulated MSCs（γMSCs） are not fully understood.MSC-derived microvesicles（MSC-MVs） have been viewed as potential pivotal mediators of the immunosuppressive effects of MSCs.Moreover,micro RNAs（miR NAs） are important regulators of immunological processes and can be shuttled from cell to cell by MVs.The aim of our study was to analyze the the mi RNA expression signature of MVs derived from γMSCs（γMSC-MVs）,which may provide better understanding of the immunosuppressive property of their parent cells.Through mi RNA microarray and bioinformatics analysis,we found 62 significantly differentially expressed miR NAs（DEMs） in γMSC-MVs compared with MSC-MVs.And the potential target genes and signaling pathways regulated by DEMs were predicted and analyzed.Interestingly,many DEMs and predicted signaling pathways had been demonstrated to be involved in immunoregulation.Furthermore,the network between immunoregulation-related pathways and relevant DEMs was constructed.Collectively,our research on the mi RNA repertoires of γMSC-MVs not only provides new perspectives into the mechanisms underlying the enhanced immunosuppressive property of γMSCs,but also paves the way to clinical application of these potent organelles in the future.

Differential Expression of APOEGene in Porcine Fetal Fibroblast Cells

[Objective]The aim was to study the differential expression ofAPOEgene in different generations of porcine fetal fibroblasts cells.[Method]The first,tenth,fifteenth,twentieth,twenty-fifth,fiftieth generations of porcine fetal fibroblast cells,which were normally grown and passed,were collected before total RNA was extracted respectively.The expression ofAPOEgene in different generations of porcine fetal fibro-blast cells was detected by RT-PCR technique.[Result]The expression level of porcine APOE mRNA in the first generation of porcine fetal fi-broblast cells was the highest,and then it gradually decreased with the increase of cell generations and was the lowest in the fiftieth generation.[Conclusion]The expression ofAPOEgene had the selective trend in different generations of porcine fetal fibroblast cells.

Construction of retroviral vectors to induce a strong expression of human interferon-β gene in human hepatoma cells

Establishing the hepatoma cell-specific expression of human interferon gene mediated by retroviral vectors. Methods: Human interferon-β complementary DNA (IFN-β cDNA) was inserted into polylinker site of pMNSM retroviral vector to construct recombinant retroviral vector pMNSIFNB, where the transcription of IFN-β gene was driven by SV40 early region promoter, and MNAIFNB, where the transcription of IFN-β gene was driven by SV40 early region promoter regulated by α-fetoprotein enhancer. The retroviral constructs were respectively introduced into PA317 amphotropic packaging cells by means of lipofectamine mediated gene transfer procedure. The plasmids transfection efficiency was among (4-25)ｘ103 colonies/μg DNA/106 PA317 cells. The retrovirus infection efficiency was among (4. 5-500)ｘ104 Colony Forming Units (CFU)/ml. The recombinant retroviruses were used to infect human hepatoma cells, renal cell carcinoma cells and melanoma cell lines in the presence of 4 μg/ml polybrene. Results: Dot hybridization of total RNA from the neomycin resistant colonies and interferon expression assay indicated that human α-fetoprotein enhancer induced efficient and specific transcription and expression of IFN-β gene driven by the promoter of different origin in human hepatoma cells by which α-fetoprotein was highly produced. Conclusion: Cis-active element of α-fetoprotein gene can drive IFN-β gene specifically expressed in human hepatoma cells, which presents some valuable materials for the hepatoma-specific immune gene therapy.

Relationship between Phenotypic Changes of Dendritic Cell Subsets and the Onset of Plateau Phase during Intermittent Interferon Therapy in Patients with CHB

Objective This study aimed to evaluate whether the onset of the plateau phase of slow hepatitis B surface antigen decline in patients with chronic hepatitis B treated with intermittent interferon therapy is related to the frequency of dendritic cell subsets and expression of the costimulatory molecules CD40,CD80,CD83,and CD86.Method This was a cross-sectional study in which patients were divided into a natural history group(namely NH group),a long-term oral nucleoside analogs treatment group(namely NA group),and a plateau-arriving group(namely P group).The percentage of plasmacytoid dendritic cell and myeloid dendritic cell subsets in peripheral blood lymphocytes and monocytes and the mean fluorescence intensity of their surface costimulatory molecules were detected using a flow cytometer.Results In total,143 patients were enrolled(NH group,n=49;NA group,n=47;P group,n=47).The results demonstrated that CD141/CD1c double negative myeloid dendritic cell(DNmDC)/lymphocytes and monocytes(%)in P group(0.041[0.024,0.069])was significantly lower than that in NH group(0.270[0.135,0.407])and NA group(0.273[0.150,0.443]),and CD86 mean fluorescence intensity of DNmDCs in P group(1832.0[1484.0,2793.0])was significantly lower than that in NH group(4316.0[2958.0,5169.0])and NA group(3299.0[2534.0,4371.0]),Adjusted P all<0.001.Conclusion Reduced DNmDCs and impaired maturation may be associated with the onset of the plateau phase during intermittent interferon therapy in patients with chronic hepatitis B.

Game-changing insights on vertebral skeletal stem cells in bone metastasis and therapeutic horizons

Greenblatt and his team have unveiled vertebral skeletal stem cells(vSSCs)as a critical player in the landscape of bone metastasis.This commentary delves into the transformative discoveries surrounding vSSCs,emphasizing their distinct role in bone metastasis compared to other stem cell lineages.We illuminate the unique properties and functions of vSSCs,which may account for the elevated susceptibility of vertebral bones to metastatic invasion.Furthermore,we explore the exciting therapeutic horizons opened by this newfound understanding.These include potential interventions targeting vSSCs,modulation of associated signaling pathways,and broader implications for the treatment and management of bone metastasis.By shedding light on these game-changing insights,we hope to pave the way for novel strategies that could revolutionize the prognosis and treatment landscape for cancer patients with metastatic bone disease.

EFFECT OF rhTGF-β1 AND rhGM-CSF ON RECEPTOR EXPRESSIONS IN J6-1 AND J6-2 LEUKEMIC CELLS

ABC immunoperoxidase was used to test the effects of rhTGF-β1 and rhGM-CSF on receptor expressions in J6-1 and J6-2 leukemic cell lines. Computer assisted image analysis system was introduced to evaluate positive index of time-and dose-dependent specimens. The expression of c-kit was elevated both in positive rate and positive index by TGF-01 in both time- and dose-dependent manners. Ing/ml rhTGF-β1 simultaneously enhanced the expression of c-fms and PDGF-R which is not detected in 50 ng / ml GM-CSF treatment. Endoglin was down-regulated after TGF-β treatment and up-regulated in J6-2 cells after GM-CSF treatment, c-kit Expression was elevated by TGF-β in J6-1 cells while decreased by both in J6-2 cells.

Effect of Chuanzhifang component (ZGC) on macrophage inflammatory injury based on whole gene expression profile

Objective: The effect of Chuanzhi Fang (ZGC) on the whole genome expression profile of RAW264.7 cells activated by lipopolysaccharide (LPS) was analyzed, and to explore the possible mechanism of action and core target of this formula on macrophage inflammatory injury at the overall level. Methods: A model of LPS-induced inflammation in RAW264.7 cells was constructed, and the effect of ZGC intervention on the genome-wide expression of inflammatory macrophages 3was examined by gene microarray technology, GO/KEGG enrichment analysis was performed for significantly differentially expressed genes among each group. Results: The results of genome-wide expression profiling microarray analysis showed that the ZGC intervention group upregulated the expression of 5 genes including C4bp and inhibited the expression of 22 genes including Mgat3, Psma6, and Siglecg relative to the LPS model group. KEGG signaling pathway analysis results showed that ZGC mainly acted through cytokine receptor interaction and the C-type lectin receptor signaling pathway. Conclusion: ZGC can interfere with the abnormal expression of 27 genes in inflammatory macrophages, and the related genes may exert corresponding anti-inflammatory effects by affecting cytokine receptor interactions, C-type lectin receptor signaling pathway, and TLR4/ NF-κB signaling pathway.

Cytokine changes and embryo attachment in mouse endometrial cells following treated with peripheral blood mononuclear cells(PBMCs)expressing ectopic hCG,and hCG-activated PBMCs

Objective:To compare the effect of human chorionic gonadotropin(hCG)-producing peripheral blood mononuclear cells(PBMCs)and PBMCs activated by hCG in vitro and expressions of related immune genes in mouse implantation.Methods:hCG-producing PBMCs(transfected PBMC)and PBMCs activated by hCG in vitro were introduced into isolated mouse endometrial cells,while cell cultures were divided into four groups:the control,PBMC,transfected,and activated PBMC groups.The expression of studied genes(IL-1β,IL-6,Lif,and Vegf)was evaluated and blastocyst attachment on the cocultured cells(isolated endometrial cells and PBMC cells)was monitored in all four groups.Results:Data showed that expression decreased in the PBMC group compared to the treated PBMC(transfected and activated PBMCs)and increased in transfected PBMC compared to the activated PBMC.Attachment and migration of blastocysts were dramatically enhanced in the transfected PBMC group compared to the activated PBMC group(P<0.05).Conclusions:Use of hCG-producing PBMCs(transfected PBMC)has more influence on endometrial receptivity.

Expression of germ cell nuclear factor in mouse germ cells and sperm during postnatal period

Aim: To assess the spatial and temporal expression of germ cell nuclear factor (GCNF) in male mouse germ cells during postnatal development and in sperm before and after capacitation. Methods: The indirect immun-ofluorescence method with anti-GCNF antiserum was used to investigate the GCNF expression in mice at day 8, 10, 14, 17, 20, 28, 35, 70, and 420 after birth and in sperm before and after capacitation. Results: With the proceeding of spermatogenesis, GCNF was first detected in the nuclei of spermatogonia and a few early stage primary sperma-tocytes at day 8, which was increased gradually at day 10 to 14 inclusive. From day 17 to day 20, the GCNF was concentrated in round spermatids, while both spermatogonia and early stage primary spermatocytes became GCNF negative. From day 28 until day 420, strong GCNF expression was shown in round spermatids and pachytene spermatocytes, while spermatogonia, early primary spermatocytes and elongating spermatids were all GCNF negative. In addition, it was also found that GCNF was localized on the acrosomal cap region of spermatozoa and there was a big change in GCNF expression during capacitation, from 98 % GCNF positive before capacitation to about 20 % positive following capacitation. The localization of GCNF in caput and cauda spermatozoa was similar. Conclusion: GCNF may play important roles in spermatogenesis, capacitation and fertilization.

Cloning of the non-structural gene 3 of hepatitis C virus and its inducible expression in cultured cells

AIM To study the inducible expression of hepatitis C virus ns3 gene (HCV ns3) in eukaryotic cells.METHODS The ns3 gene was obtained from plasmid pBns3 by polymerase chain reaction and inserted into the cloning vector pGEM-T. Then, the ns3 was subcloned into the vector pMSG to generate dexamethasone (DM)-inducible expression plasmid pMSG-ns3. CHO cells were transfected by pMSG-ns3 using calcium phosphate precipitation method and cultivated for 12 h-24 h. The transfected cells were induced with DM and the transient expression of NS3 protein was analyzed by ELISA and Western-blot methods.RESULTS After treated with 3×10-8mol/ L DM, the expression of NS3 was observed in the transfected CHO cells. A slightly higher level of NS3 was shown along with the time of DM treatment.CONCLUSION The inducible expressing vector pMSG-ns3 might be helpful for further studies of the characteristics of the ns3 gene in vivo.

Expression of the Human Growth Hormone Genein Insect Cells

The insect cellvirus (AcNPV) system was used to express heterologous protein. The recombinant transfer vector pVL1393/hGH was reconstructed in which the human Growth Hormone (hGH) gene was inserted under the control of the polyhedron gene promoter. The Spodoptera frugiperda (Sf9) cells was cotransfected with the plasmid DNA containing hGH gene and wildtype AcNPV DNA. The hGH gene was transferred to the AcNPV genome DNA through homologous recombination, and the recombinant virus rAcVhGH was obtained by multiple plaque purification. The high level of production of hGH (40 μg/mL) in supernatant of the infected monolayer culture was determined by immunochemiluminescent assay.

Differences in phenotype and gene expression of prostate stromal cells from patients of varying ages and their influence on tumour formation by prostate epithelial cells

Prostate cancer （PCa） is an age-related disease, and the stromal microenvironment plays an important role in prostatic malignant progression. However, the differences in prostate stromal cells present in young and old tissue are still obscure. We established primary cultured stromal cells from normal prostatic peripheral zone （PZ） of donors of varying ages and found that cultured stromal cells from old donors （PZ-old） were more enlarged and polygonal than those from young donors （PZ-young）. Furthermore, based on immunocytochemical and ultrastructural analysis, the components of stromal cells changed from a majority of fibroblasts to a mixture of fibroblasts and myofibroblasts with increasing donor age. Using a three-dimensional in vitro culture system, we found that PZ-old stromal cells could enhance the proliferation, migration and invasion of cocultured benign BPH-1 and PC-3 cells. Using an in vivo tissue recombination system, we also found that PZ-old stromal cells are more effective than PZ-young cells in promoting tumour formation by BPH-1 cells of high passage（〉100） and PC-3 cells. To probe the possible mechanism of these effects, we performed cDNA microarray analysis and profiled 509 upregulated genes and 188 downregulated genes in PZ-old cells. Among the changed genes, we found genes coding for a subset of paracrine factors that are capable of influencing adjacent epithelial cells; these include hepatocyte growth factor （HGF）, fibroblast growth factor 5 （FGF5）, insulin-like growth factor 2 （IGF2）, insulin-like growth factor-binding protein 4 （IGFBP4）, IGFBP5 and matrix metallopeptidase 1 （MMP1）. Changes in the expression of these genes were further confirmed by quantitative real-time polymerase chain reaction （PCR）, Western blotting and enzyme-linked immunosorbent assays. Overall, our findings indicate that stromal cells from prostate PZ of old donors are more active than similar cells from young donors in promoting the malignant process of adjacent epithelial cells. This finding hints at a new potential strategy for the prevention of PCa.