| Background and ObjectiveCervical cancer is a malignant tumor threatening the health of females.Currently,clinical treatments include surgery,chemotherapy,radiotherapy,and biotherapy.Although the treatments showed definite effects,they have various side-effects and a highrecurrence rate (about35%).Various prophylactic vaccines that were developed based onHPV still cannot meet clinical needs due to immune type,vaccination time,long-term effectsand other factors.Therefore,the mechanism of cervical cancer should be further studied soas to improve the existing treatment strategies and develop more new effective treatmentsthat are extremely important in reducing the mortality rate of cervical cancer.Persistent HPV infection is definite etiological factor for cervical cancer,the formationof human malignan tumour is not only malignant behavior,but also related with theantitumor immune surveillance function of body. Dendritic cells (DCs), specializedantigen-presenting cells (APCs), have strong functional antigen presentation capability.Inrecognition of partial risk antigen,immature DC positioning in the peripheral tissues willmigrate to draining lymph nodes.Mature DC can active the immune response, thus,thedifferentiation and maturation of DC is the key to the specific antitumor immunity.Thestudy found,the number of mDC was significantly down-regulated in cervical cancer;invitro,differention of DC was significantly down-regulated by SiHa cells(cervical cancel cellline,HPV16+) culture supernatant.The research shows that,differentiation anddevelopmental condition of DC were abnormal during the occurrence and development ofcervical cancer.Differentiation of DC involves complex physiological regulatory mechanisms. Thereare many factors such as cytokines of local microenvironment and various kinds of activesubstance that control DC differentiation. Recent studies found that pigmented epithelium-derived factor (PEDF)is widely distributed in mammals, which is anendogenous glycoprotein expressed abnormally in a series of tumor tissues (includingcervical squamous cell carcinoma). It has a variety of biological characteristics and canpromote recruitment and activation of macrophage which originated from the same cells asDC. Practical tests have shown that PEDF can significantly increase the migrationcapability of mouse bone marrow-derived DC. That suggested that the important activesubstrate PEDF in the cervix is an important regulation factor for the differentiation andmaturation of DC, and the loss of PEDF in the cancer tissue is an important factor for theinhibition of the differentiation and maturation of DC. In view of the close relationshipbetween PEDF and DC, we explored the regulation of PEDF on the DC biological functionsuch as phenotypic differentiation, synthesis and secretion and antigen presentation byobserving the PEDF expression of the local tissues of invasive cervical carcinoma and thedistribution of DC, and further cleared the effect of PEDF in the immune escape of cervicalcancer, might reverse the DC function abnormity, so as to provide a preliminaryexperimental basis for rebuilding the immune surveillance efficiency of patients andimprove the local anti-tumor immunity, and provide a new therapeutic strategy for theclinical treatment of cervical cancer.Materials and Methods:I.The PEDF expression and DC distribution in cervical cancer: Normal cervical tissueswere used as a control group (10cases, from patients with uterine fibroids),the cervicallesions of patients with invasive cervical cancer in different stages were also obtained (Ib10cases, stage II10, stage III6cases). PEDF expressions were determined by Western blot,DC distribution of cervical lesions were detected by immunofluorescence.II. The effect of PEDF on the differentiation and antigen-presenting function ofbone marrow-derived dendritic cells(BMDC): The bone marrows from normal6-8weeksC57BL/6mice’ femur and tibia were obtained. Furthermore, the erythrocytes wereremoved by erythrocyte-cracking solution. In the presence of recombinant mousegramulocyre-macrophage (rmGM-CSF) and recombinant mouse interleukin-4(rmIL-4),thebone marrows were cultured in vitro to induce DCs. DCs grouping were as follows: GroupA: DCs+PEDF (50ng/mL); Group B:DCs+PEDF (100ng/mL);Group C:DCs+PEDF(200ng/mL);Group D:DCs+LPS (1ug/mL);Group E:DCs+1640.The cells were under morphological observation. The changes of DCs expression, CD11c, CD80and CD86ineach group were determined by Flow CytoMetry(FCM);The stimulating capacity of DCwere determined by mixed lymphocyte reaction(MLR); The IL-12secretion levels weredetected by enzyme-linked immunosorbent assay.Results:I.The PEDF expression and DC distribution in cervical cancer:1. PEDF expression in invasive cervical squamous cell carcinoma: The PEDF proteinlevels of diseased tissue in patients with invasive cervical squamous cervical lesions werelower than that in normal cervical tissue (P <0.05).2. The local CD1a (+) cell density and CD83(+) cell density of invasive cervicalsquamous cell carcinoma were both decreased (P <0.05).II. The effect of PEDF on the differentiation and antigen-presenting function ofbone marrow-derived dendritic cells(BMDC).1.Morphological observation: After culturing a GM-CSF-induced mouse BMDC for5days, there were few burr-like processes on the cell surface, cells aggregated, then becamecolony-like and grew with adherence. After adding different concentrations ofPEDF(50ng/ml、100ng/ml、200ng/ml) and LPS(positive control,1ug/ml), the process on thecell surface increased and the cell colony enlarged.2. Phenotypic differentiation: After culturing GM-CSF induced mouse BMDC indifferent concentrations of PEDF(50ng/ml、100ng/ml、200ng/ml),flow cytometry detectionshowed that as cell surface molecules CD11c expression levels were significantlyincreased(PEDF50、100、200ng/ml，92.80±3.81%、87.43±7.98%、92.53±3.13%，P<0.05),and the difference was statistically significant;CD80expression levels weresignificantly increased(PEDF50、100、200ng/ml，92.77±3.50%、94.10±2.76%、93.30±3.22%，P<0.05),and the difference was statistically significant; CD86expression levelswere significantly increased(PEDF50、100、200ng/ml，77.53±2.94%、78.27±1.99%、79.13±6.10%，P<0.05), and the difference was statistically significant.3. Mixed lymphocyte reaction(MLR): After culturing a GM-CSF-induced mouseBMDC in different concentrations of PEDF(50ng/ml、100ng/ml、200ng/ml), the stimulationindex(SI) expression was significantly increased(PEDF50、100、200ng/ml，1.44±0.11、1.61±0.40、1.59±0.29，P<0.05),and the difference was statistically significant. 4. IL-12secretion levels: After culturing a GM-CSF-induced mouse BMDC indifferent concentrations of PEDF(50ng/ml、100ng/ml、200ng/ml), the IL-12secretion levelswere significantly increased(PEDF50、100、200ng/ml，222.27±8.01、227.47±4.58、214.83±12.64pg/mL，P<0.05),and the difference was statistically significant.Conclusions:1.The local PEDF expression level of invasive cervical squamous cell carcinoma wassignificantly reduced, DC distribution density decreased, and the maturity level of thedifferentiation also decreased, this suggests that the level of the PEDF expression may berelated to the local DC dysfunction in cervical carcinoma tissue.2. PEDF can significantly increase the CD11c, CD80, CD86expression levels on themouse BMDC surface and increased the Il-12secretion and activate the T lymphocyteproliferation, it can be used as the positive regulator for the immune function of DCs.3. the loss of PEDF can induce DC immune dysfunctional, which is one of the cruciallinks for the immune escape of cervical cancer. Its regulating mechanism still needs furtherstudy. This can provide a preliminary experimental basis for the reverse of DC functionabnormity and rebuild the immune surveillance efficiency of patients, so as to improve thelocal anti-tumor immunity. It provides a new therapeutic strategy for the clinical treatmentof cervical cancer. |
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