10铜低密度聚乙烯纳米复合材料对大鼠子宫内膜血管再生影响的相关性研究

1 5 10 15 20 25 30 35 40 45 50 5515101520Front. Med. China 2007, 1(4): 1–4DOI 10.1007/s11684-007-0000-0Correlative investigation of copper/low-density polyethylene nanocomposite on the endometrial angiogenesis in ratsLI Jianxiong1, MS, LIU Zilong1, BS, LI Shuang2, MD, XIE Changsheng 3, PhD, DUAN Yonggang1, MS,YU Jing1, MS, ZHU Changhong ( )1, PhD MD1 Family Planning Research Institute, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China2 Cancer Biology Research Center, Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College,Huazhong University of Science and Technology, Wuhan 430030, China3 Department of Materials Science and Engineering, Huazhong University of Science and Technology, Wuhan 430074, China© Higher Education Press and Springer-Verlag 2007E-mail: zhuch@21 5 10 15 20 25 30 35 40 45 50 551510152025303540455055 contribute to satisfactory contraceptive efficacy with slighterside effects.2 Materials and methods2.1 Materials2.1.1 AnimalsSexually mature female SD rats, weighing (220P20) g,were obtained from the Experiment Animal Center of TongjiMedical College, Huazhong University of Science and Tech-nology. Rats were reared with standard conditions (12 h light/12 h dark cycle, (25P3)°C, 35%–60% relative humidity),and rat feed and tap water were available ad libitum.2.1.2 IUD component materialsThe IUD component materials, provided by the department ofmaterials science and engineering in Huazhong University ofScience and Technology, were divided into three kinds: bulkcopper, LDPE and nano-Cu/LDPE (5–10 µg/220 mm2/dayand 11–20 µg/220 mm2/day, respectively). For each material,the surface area of each sample was 4 mm2.2.2 Methods2.2.1 Animal treatmentOne hundred sexually mature female SD rats were recruitedand randomly divided into five groups: sham-operationgroups (SO group, n =20), bulk copper groups (Cu group,n =20), LDPE groups (n =20), nano-Cu/LDPE groups I(5–10 µg/220 mm2/day, n = 20) and II (11–20 µg/220 mm2/day, n =20). Rats in Cu, LDPE and nano-Cu/LDPE groupswere anesthetized with 10% chloral hydrate (3mg/kg, i.p),and then each corresponding material was inserted into thecaudal portion of unilateral uterine horn and secured to theuterine wall via operations including laparotomy and uterot-omy. Rats in SO groups were treated with the same operationsexcept for inserting materials.2.2.2 ImmunohistochemistryParaffinembedded tissue sections were deparaffinized andrehydrated. Slides were treated with 3%H2O2 for 10 min.After incubation, the slides were washed in PBS and incu-bated with 3% bovine serum for 30 min at room temperatureto reduce nonspecific binding, and then the slides were incu-bated with goat-anti-rat anti- Ang-2 polyclonal antibody(Santa Cruze, USA, 1 : 150 dilution), rabbit-anti-rat anti-Tie-2polyclonal antibody (Santa Cruze, USA, 1 : 50 dilution) andrabbit-anti-rat anti-CD34 polyclonal antibody (Boster, China,1 : 150 dilution) for 1 h at 37°C, respectively. After the slideswere treated with the second biotinylated antibody, PBS andS-P agents (Zhongshan, China), the slides were visualizedwith 0.1% diaminobenzidine solution and then counterstainedwith hematoxylin. The slides were quickly dehydrated byethanol, lucidificated by dimethyl benzene and mounted withneutral gummi.2.2.3 Image analysisFive photos for each slide were taken with the imagingsystem of the Olympus multifunction microscope at the sameconditions of section, dyeing and illumination. By use ofcell measure procedures in the HMIAS-2003 high-definitioncolor medical image analysis system (Tongji Qianpin Image,China), the expression of Ang-2, Tie-2 in different groups wasanalyzed with the average grey scales in eyeshot, and then themean and standard in each group were calculated.2.2.4 Microvessel densityMicrovascular endothelial cells were marked with CD34, andthe stained microvessels were calculated in five eyeshots(x200), and then the mean and standard in each group wereanalyzed as MVD.2.3 Statistical analysisSPSS 12.0 statistical software was used to perform statisticalanalysis. All the values were expressed as x r P s. A one-factorANOV A and t test were used for statistical evaluation. P<0.05was considered statistically significant.3 Results3.1 Ang-2 and Tie-2 levelsThe positive stained cells were full of brown-yellow Ang-2and Tie-2 proteins in the cytoplasm, respectively. Comparedwith those in the SO group, the expression of Ang-2 andTie-2 in all the experimental groups was obviously increasedon the 30th day after insertion, and these parameters in nano-Cu/LDPE groups, except for Ang-2 level in nano-Cu/LDPEgroup II, were significantly lower than those in Cu group(P<0.05). Ang-2 and Tie-2 levels were still higher in Cugroup and LDPE group (P<0.05), but there was no differ-ence of Ang-2 and Tie-2 levels between nano-Cu/LDPEgroups and SO groups on the 180th day after insertion(P>0.05) (Figs. 1 and 2).3.2 CD34 levels and MVDThe expression of CD34 was shown by brown stainingand located in the cytoplasm of vascular endothelial cells.Compared with those in the SO group, the significant increasesof MVD were observed on the 30th day and the 180th dayafter the insertion of the bulk copper(P<0.05). MVD countswere not significantly different before and after the insertionof nano-Cu/LDPE (P>0.05) (Fig. 3).3151015202530354045505515101520253035404550554 DiscussionThe contraceptive efficacy of Cu-IUDs has increasedobviously in comparison to that of the conventional IUDs;at the same time, the rates of pregnancy with IUDs havedecreased by 0.3%–1.5% [4,5]. The bioactive copper ions,released into the cavity of uterus continuously, contribute tothe satisfactory contraceptive efficacy, while the side effectsof copper corrosion results in undesired rates. Large-scaleepidemiologic studies indicate that such improvements,which were mainly involved in the addition of antihemor-rhagic into the device or the change of the size and the shapeof the device [6], have not solved the inherent disadvantagesof Cu-IUD. The majority of side effects attributed to thechanges of endometrial microenvironment take place at theinitial stage after Cu-IUD insertion such as inter menstrualbleeding or spotting, and they are most likely relevant tothe burst release of copper ions. On the one hand, the trans-formation ratio of copper ions in bulk copper is relativelylow reaching 33% [7]. On the other hand, diverse corrosiveproducts participate in the formation of deposite coating andmay be related to the side effects, in addition to Cu2+, suchas calcite (CaCO3), calcium phosphate, cuprite (Cu2O) andcopper hydroxide [Cu (OH)2], etc, [8,9]. And the fragmenta-tion of copper wires or sleeves and deposits on them leads tothe unexpected failure of Cu-IUD. Therefore, it is necessaryto control the release process of copper ions, and to alter thecopper corrosion patterns.The copper/low-density polyethylene nanocomposites(nano-Cu/LDPE) with various mass fractions of coppernanoparticles have been prepared successfully, and the prepa-ration was previously described as Xie [3] (Fig. 4). Briefly,the potential IUD component materials, which consisted ofcopper nanoparticles instead of bulk copper such as coppersleeves or wires, were manufactured by using physicochemi-cal methods to combine high quality copper nanoparticles(Patent No.ZL98113626.5) and LDPE powders. Not only thespecial effects of cupric ions but also the controlled releasetechnology contributes to the nano-Cu/LDPE preparation. Ithas been proved that the burst release behavior of copper ionsat the initial stage can be inhibited and that the samplesdisplay near zero-order releases after a month of incubation[3]. No compounds depositing on the composite surface faci-li t ate the corrosion of copper nanoparticles deeply insidethe composite and the outward diffusion of cupric ions. Ourother research previously suggested that the nano-Cu/LDPEexhibited satisfactory contraceptive effectiveness, which wassimilar to that of the conventional bulk copper materials [10].In this study, it has been revealed that the significant increases Note: As compared with the SO group, *P<0.05; As comparedwith the Cu group, ∆P<0.05Fig. 1Grey values of Ang-2 in different groups on the 30th dayand the 180th day after insertionNote: As compared with the SO group, *P<0.05; As comparedwith the Cu group, ∆P<0.05Fig. 2 Grey values of Tie-2 in different groups on the 30th dayand the 180th day after insertionNote: As compared with the SO group, *P<0.05Fig. 3 MVD counts in different groups on the 30th day and the180th day after insertion415101520253035404550551510152025303540455055of MVD were observed after the insertion of the bulk copper (P <0.05). MVD counts were not significantly different before and after the insertion of nano-Cu/LDPE (P >0.05). It has been suggested that the effects of nano-Cu/LDPE on vascular endothelial cells in endometria was rather slighter and more transient than those of conventional bulk copper. The transiently and slightly high release behavior of nano-Cu/LDPE due to that partial copper nanoparticles attached to surface of the composites, without diffusion through pore canals, can directly react with endometria in a short time, but it is not strong enough to result in obvious increases of MVD counts.Ang-2 has been considered as one of the most effective and specific modulators among numerous regulatory factors of endometrial angiogenesis. Ang-2, believed to antagonize the stabilizing action of Ang-1, belongs to a novel family of angiogenic factors and binds with similar affinity to the endothelial cell tyrosine kinase receptor Tie-2 [11,12]. In the presence of VEGF, vessel destabilization by Ang-2 has been hypothesized to induce an angiogenic response; however, in the absence of VEGF, Ang-2 leads to vessel regression [12,13]. The burst release of copper ions and the focal hypoxia caused by the oppressive effects of IUDs were associated with the increasing expression of VEGF in endometria after insertion [14]. The Ang-1/Tie-2 signal transduction and antagonizing the stabilizing action of Ang-1, the Ang-2/Tie-2 system inhibits the interaction between endothelial cells and peritubular cells, enhances vessel permeability, and makes the vessel structure unstable. According to the results in this study, compared with those of traditional bulk copper, Ang-2/Tie-2 levels in nano-Cu/LDPE groups were slightly increased (P <0.05).From the above results, we can safely conclude that the effects of nano-Cu/LDPE on the levels of angiogenesis related factors, such as Ang-2 and Tie-2, were slighter in comparison to those of the bulk copper, and the release of vasoactive sub-stances and the changes of the endometrial microenvironment could be reduced by using nano-Cu/LDPE. It is presumed that the side effects after insertion, for example uterus abnormalbleeding, may be relieved by weakening the burst release behavior of copper ions and no compounds deposit on the composite surface. Therefore, nano-Cu/LDPE may be a potential substitution for the conventional IUDs in the near future due to the satisfactory contraceptive efficacy and slight side effects on the endometrial microenvironment.Acknowledgements The authors gratefully acknowledge the financial support of Key Technologies Research and Development Programme of the National Tenth Five Years Plan (NO.2006BA103B01).References1. Holland M K, White I G. Heavy metals and spermatozoa. III. Thetoxicity of copper ions for spermatozoa. Contraception, 1988, 38(6): 685–6952. Araya R, Gomez-Mora H, Vera R, Bastidas J M. Human sperma-tozoa motility analysis in a Ringer’s solution containing cupric ions. Contraception, 2003, 67(1): 161–1633. Cai S Z, Xia X P, Xie C S. Corrosion behavior of copper/LDPEnanocomposites in simulated uterine solution. Biomaterials, 2005, 26(15): 2671–26764. 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