Photodynamic Therapy Using a Diode Laser with Mono-l-aspartyl Chlorin e6 for Implanted Fibrosarcoma in Mice

We have developed a new high-power red (664 nm) laser diode system for photodynamic therapy (PDT) with mono-L-aspartyl chlorin e6 (NPe6). Meth-A fibrosarcoma cells (1X10⁶) were implanted subcutaneously in the right hind leg of 4-week-old BALB/c female mice. One week later, diode laser irradiation was applied 5 h after the intravenous administration of NPe6 to each tumor-bearing mouse. In the first study, the time course of intratumor temperature increase during PDT was measured by using a 23-guage thermocouple hypodermic needle at a depth of 2 mm from the tumor surface. In the second study, 6 groups of 10 to 17 tumor-bearing mice were treated with the diode laser 5 h after intravenous administration of NPe6 at the dose of 1.25, 2.5, 5.0 or 7.5 mg/kg i.v. per mouse. Total photoirradiation ranged from 0 to 150 J/cm² and the dose rate was adjusted to 100 mW/cm². Percentages of cures were determined from numbers of mice apparently disease-free 50 days after treatment. The results showed that this diode laser is effective in PDT of implanted fibrosarcoma after NPe6 administration. It also confirmed that the therapeutic effects of PDT were not due to hyperthermia. Moreover, the diode laser beam was demonstrated by CCD technology to be uniform in intensity throughout the photoirradiated field.     

Effects of Photodynamic Therapy Using Mono-l-aspartyl Chlorin e6 on Vessels and Its Contribution to the Antitumor Effect

The effect of photodynamic therapy (PDT) on the vascular system has a significant role in tumor tissue destruction. We investigated the contribution of vascular damage to the antitumor effects of PDT and analyzed the quantitative vascular changes after PDT. Fibrosarcoma-bearing BALB/c male mice were injected with mono-L-aspartyl chlorin e6 (NPe6) at a dose of 0.25, 5 or 15 mg/kg, and photoradiation was performed with a diode laser 10 min, 2 h or 24 h after injection, respectively. Ten minutes after injection of 0.25 mg/kg, NPe6 was found to be present only in plasma, while at 2 h after injection of 5 mg/kg it was present in both plasma and tumor, and 24 h after injection of 15 mg/kg it was present only in the tumor. The antitumor effects observed in the 5 mg/ kg-2 h and 0.25 mg/kg-10 min groups were virtually the same, whereas the effect in the 15 mg/kg- 24 h group was weaker. The damage to the tumor vasculature and tumor cells in the 15 mg/kg-24 h group occurred later than under the other conditions, and vascular damage in the tumor-surrounding tissue was also less marked even 24 h after PDT. These results suggested that the plasma NPe6 concentration during laser irradiation contributed more than the tumor NPe6 concentration to the antitumor effect, and that the minimal damage to blood vessels around the tumor at the low plasma NPe6 concentration may be one reason for the failure to obtain a marked antitumor effect.     

Effects of photodynamic therapy using mono-L-aspartyl chlorin e6 on vessels and its contribution to the antitumor effect.

The effect of photodynamic therapy (PDT) on the vascular system has a significant role in tumor tissue destruction. We investigated the contribution of vascular damage to the antitumor effects of PDT and analyzed the quantitative vascular changes after PDT. Fibrosarcoma-bearing BALB / c male mice were injected with mono-L-aspartyl chlorin e6 (NPe6) at a dose of 0.25, 5 or 15 mg / kg, and photoradiation was performed with a diode laser 10 min, 2 h or 24 h after injection, respectively. Ten minutes after injection of 0. 25 mg / kg, NPe6 was found to be present only in plasma, while at 2 h after injection of 5 mg / kg it was present in both plasma and tumor, and 24 h after injection of 15 mg / kg it was present only in the tumor. The antitumor effects observed in the 5 mg / kg-2 h and 0. 25 mg / kg-10 min groups were virtually the same, whereas the effect in the 15 mg / kg-24 h group was weaker. The damage to the tumor vasculature and tumor cells in the 15 mg / kg-24 h group occurred later than under the other conditions, and vascular damage in the tumor-surrounding tissue was also less marked even 24 h after PDT. These results suggested that the plasma NPe6 concentration during laser irradiation contributed more than the tumor NPe6 concentration to the antitumor effect, and that the minimal damage to blood vessels around the tumor at the low plasma NPe6 concentration may be one reason for the failure to obtain a marked antitumor effect.     

High expression of GADD-45alpha and VEGF induced tumor recurrence via upregulation of IL-2 after photodynamic therapy using NPe6

NPe6 is a novel second-generation photosensitizer used for photodynamic therapy (PDT). PDT using NPe6 and diode laser (664 nm) induces cell death, inflammatory reactions, immunological responses and damage to the microvasculature. In this study, we evaluated the influence of the immunological responses and of enhanced angiogenesis on the anti-tumor effect of NPe6-PDT using cytokine-overexpressing Lewis lung carcinoma (LLC), LLC-IL-2 cells both in vitro and in vivo. We showed by DNA microarray analysis in vitro that IL-2 and GADD-45alpha (growth arrest and DNA damage 45 alpha) mRNA expressions were induced by 3 h after NPe6-PDT applied at a dose killing 90% of the cells (LD90). IL-2-overexpressing cells (LLC/IL-2 cells) were resistant to the loss of clonogenicity as compared to the parental LLC cells in vitro. Furthermore, in female C57BL/6 mice, NPe6-PDT produced a cure rate of 66.7% in LLC tumors, whereas the cure rate was only 16.6% in LLC/IL-2 tumors, and overexpression of IL-2 caused failure of NPe6-PDT, with tumor recurrence, in vivo. These results suggest that IL-2 expression may play an unfavorable role in attenuation of the antitumor effect of NPe6-PDT. It has been reported that the expression of vascular endothelial growth factor (VEGF), in particular, may cause tumor recurrence after PDT and exert unfavorable effect in relation to attenuate the anti-tumor activity of PDT. Results of immunohistochemical analysis of LLC/IL-2 tumors have revealed that the expressions of GADD-45alpha and VEGF are induced in these tumors after PDT, and in particular, 12 h after PDT, the expression levels were much higher as compared with those in the LLC tumors. The results of our studies using in vitro and in vivo models suggest that the cell death caused by PDT was inhibited by induction of GADD-45alpha expression and that tumor recurrence was promoted by the enhancement of VEGF expression mediated by IL-2 upregulation. Therefore, it is speculated that the use of an IL-2 inhibitor may improve the efficacy of NPe6-PDT.     

The effect of a synthetic fragment of human interleukin-2 on the growth and vascularization of sarcoma 180 in mice]

The effect of a modified peptide of the middle part of the human interleukin-2 molecule (C-1-6) on the growth of transplantable sarcoma 180 of mice was studied. C-1-6 injected intraperitoneally in the dose of 0.5, 5 and 50 micrograms 4 times every other day was shown to stimulate tumor growth and enhance angiogenesis induced by the tumor transplanted intracutaneously. Stimulation of tumor growth was most apparent when the agent was given late (on day 4) after transplantation. Supernatants of murine peritoneal macrophages activated by 1 microgram/ml solution of C-1-6 proved capable of stimulating tumor growth, too. It is suggested that the stimulating effect of C-1-6 on tissue regeneration be investigated.     

Metabolic properties and photosensitizing responsiveness of mono-L-aspartyl chlorin e6 in a mouse tumor model.

A mouse mammary tumor model was used to evaluate metabolic properties of the photosensitizer mono-L-aspartyl chlorin e6 (NPe6) and to determine the optimal time interval between drug administration and light treatment for effective photodynamic therapy (PDT). Photosensitizer metabolism was evaluated by comparing tissue distribution patterns of NPe6 having 14C atoms positioned on either the tetrapyrrole ring or on the aspartyl residue. High performance liquid chromatographic analysis of photosensitizer extracted from tumor tissue was also obtained as a function of time after drug administration. NPe6 distribution in tissue samples and pharmacological calculations of area under the curve were similar for both forms of [14]NPe6. Likewise, metabolic contaminants of NPe6 were not detected by high performance liquid chromatographic analysis following extraction of the photosensitizer from tumor tissue. Maximal in vivo PDT effectiveness was achieved when light treatments were started within 2 h of drug injection. PDT effectiveness was decreased by 50% when light treatments were initiated 6 h after drug injection and was abolished with a 12-h interval between NPe6 injection and light exposure. Responsiveness to NPe6-mediated PDT was correlated with photosensitizer levels in the plasma but not in tumor tissue. These results show that NPe6 was not metabolized following in vivo administration and that the responsiveness of NPe6 mediated PDT was associated with vascular clearance of the photosensitizer.     

Tissue distribution and photosensitizing properties of mono-L-aspartyl chlorin e6 in a mouse tumor model

Mono-L-aspartyl chlorin e6 (NPe6) is a photosensitizer that possesses properties such as chemical purity and a major absorption band at 664 nm which are potentially exploitable for photodynamic therapy (PDT). The current investigation examined pharmacological and photosensitizing parameters of NPe6 in tumor and normal tissues in mice. [14C]NPe6 was used to obtain quantitative tissue distributions of the photosensitizer as a function of: (a) time following administration; (b) drug dose; (c) mode of drug administration; and (d) tumor size. The in vivo photosensitizing efficiency of NPe6 was compared directly to Photofrin II in experiments which evaluated tumor responses and induction of normal skin damage. Initial PDT experiments demonstrated that NPe6 was ineffective at inducing tumor cures when a 24-h time interval (between drug administration and light treatment) was used. However, PDT-induced tumor cures were obtained when NPe6 was administered 4-6 h prior to light exposure, and these NPe6-PDT treatment parameters were as effective as standard Photofrin II-mediated PDT. Interestingly, the level of PDT-induced normal skin damage was significantly greater for Photofrin II than for NPe6 at comparable drug and light doses. An analysis of pharmacological data and PDT time interval requirements suggests that plasma concentrations of NPe6 may be a more important predictive factor than tumor tissue levels of the photosensitizer for the production of PDT-mediated tumor cures. The results of this investigation indicate that NPe6 is an effective tumor photosensitizer with in vivo clearance properties that eliminate the side effect of prolonged normal skin photosensitization.     

Tumor-host interactions accompanying the growth of the G:5:113 fibrosarcoma in the mouse: possibilities for a new therapeutic approach?

The experiments were aimed at describing in detail some interactions between a solid tumor growing from subcutaneously transplanted G:5:113 fibrosarcoma cells in vivo and its mouse host. The tumor was found to elevate significantly the number of granulocytes in the peripheral blood of the host after having achieved the volume of about 1 cm³ (day 40 after transplantation). Blood plasma from fibrosarcoma-bearing mice stimulated proliferation of progenitor cells for granulocytes and macrophages (GM-CFC) in vitro and suppressed growth of G:5:113 cell population in culture. Interestingly, both effects were observable as early as week 1 when the tumor was still macroscopically invisible and unpalpable. Conditioned medium from cultures of G:5:113 fibrosarcoma cells stimulated proliferation of GM-CFC in vitro. These findings might represent a starting point for studies aimed at designing new therapeutic approaches for the treatment of fibrosarcoma.     

人肺腺癌细胞LALU血管生成过程的形态学及超微结构观察

目的 观察人肺癌细胞血管生成过程的病理形态学、超微结构特点。方法 采用人肺腺癌细胞株LALU经SICO鼠皮下移植瘤模型,用光镜和电镜动态观察不同时期肿瘤血管生成状态。结果 光镜显示,人肺腺癌移植瘤第2－10天时可分为血管生成前期和血管形成期,第20天出现肺转移灶。电镜显示,人肺腺癌移植瘤第2天出现成血管细胞;第4－10天,不成熟血管内皮细胞逐渐形成血管腔并伴有较完整的新生基底板,内皮细胞趋向成熟发展;第20天肿瘤血管内皮细胞更成熟,部分区域新生毛细血管基底板发育不或缺陷。其全过程中癌细胞突起直接与成血管细胞、血管内皮细胞及血管壁相连。结论 病理形态学及电镜的特征形态学改变,提示肿瘤血管生成与转移有关,可为肺癌的血管导向治疗提供重要依据。     

Radiosensitization of two murine fibrosarcomas with 6-thioguanine

In Vivo murine tumor experiments were carried out to determine whether 6-thioguanine (6-TG) could enhance the cytotoxic effects of radiation on tumors. The combined effects of single and fractionated x-irradiation were evaluated on the transplanted methylcholanthrene induced fibrosarcoma (Meth-A) in BALB/c mice, a moderately radioresponsive tumor and on the radiation induced fibrosarcoma (RIF) in C3H/He mice, a highly radioresistant tumor. The combined treatment of single administration of 6-TG (25 mg/kg) and of x-irradiation (20 Gy) on Meth-A tumors produced more than 90% tumor control, whereas the radiation alone resulted in less than 5% tumor control. The radiosensitizing effect by 6-TG was higher when the drug was administered either 1 to 8 hr prior to or 24 hr after x-irradiation. The dose modification factor of single dose 6-TG (10 mg/kg) is estimated to be 1.47 for Meth-A tumor and 1.25 for RIF tumor. The tumor control rates of fractionated irradiation alone and with concomitant 6-TG in Meth-A tumors were 14% and 59%, respectively. Based on the studies reported here and well documented pharmacokinetics in humans, it is suggested that combined radiation therapy and 6-TG may provide an enhanced therapeutic effect even in tumor varieties where the drug has no apparent anti-tumor activity on non-irradiated cells.     

Prevention of Hepatic and Peritoneal Metastases by the Angiogenesis Inhibitor FR-118487 after Removal of Growing Tumor in Mice

We established a mouse "primary tumor resection model" in which a transplanted tumor was resected after an orthotopic transplantation of colorectal cancer tissue to estimate the therapeutic effect of an angiogenesis inhibitor on metastasis. The angiogenesis inhibitor FR-118487 is a member of the fumagUlin family. Here, 1 mg/kg/day of FR-118487 was subcutaneously administered to nude mice for 1 week, 2 weeks, or 4 weeks through an osmotic pump. Liver metastasis developed in 7 of 9 control mice, 2 of 6 mice that underwent the tumor resection 2 weeks after transplantation (early resection), and in all 7 of the mice that underwent the tumor resection 4 weeks after transplantation (late resection). In the short treatment trial, the FR-118487 administration immediately after the early resection completely inhibited both hepatic and peritoneal metastases, whereas its administration after the late resection had no effect on liver metastasis. In the prolonged treatment trial, inhibitory effects of prolonged treatment with FR-118487 on both hepatic and peritoneal metastases after the late resection were clearly demonstrated. The mice of the resection-alone group all died within 106 days after tumor inoculation, due to metastases of colon carcinoma. In contrast, half of the mice that underwent resection and then received antiangiogenic therapy were alive at the end of the observation period (160 days after transplantation). In conclusion, the combination of surgery and subsequent antiangiogenic therapy may be useful to prevent the distant metastasis of colorectal cancer and to improve the prognosis of patients with colorectal cancer.     

人乳腺癌细胞系MCF—7原位移植血管生成超微结构观察及其相关因素表达

目的：探讨人乳腺癌细胞增殖与血管生成方式和时间的超微结构特点,明确人乳腺癌生长中VEGF、F8因子表达水平及其意义,方法：应用人乳腺癌细胞系MCF－7原位移植SCID鼠,光镜、电镜动态观察肿瘤生长与血管生成的细胞形态,结合免疫组化检测VEGF、F8因子的表达水平。结果：人乳腺癌细胞系MCF－7移植后1、2天生存靠固有组织的血管滋养生长,肿瘤组织中未见新生血管生成。肿瘤生长第3天时,肉眼可见周围血管血移植的肿瘤表面延伸,电镜下观察在 存活的癌细胞中可见血管母 细胞出现。第6天时,在癌细胞增生活跃区血管母细胞演化为血管内皮细胞,此时癌细胞群与新生的肿瘤微血管紧密相连。微血管邻旁周边可见极少量的蛋白质网架。第8－10天,内皮细胞有较丰富的线性粒体,粗面内质网增多,池内含有中等量蛋白质,血管内皮的腔面小球增多,光镜下可见肿瘤周围的血管与肿瘤新生血管相通。VEGF、F8因子的表达与肿瘤增殖,侵袭、新生血管生成有关。结论：移植初期人乳腺癌细胞何分泌血管生成因子,肿瘤周围的血管母细胞被激活,快速增殖期癌细胞可诱导血管内上细胞增生形成肿瘤新生血管,逐步演化的肿瘤新生血管与肿瘤边缘固有的血管相连通。     

Prevention of hepatic and peritoneal metastases by the angiogenesis inhibitor fr-118487 after removal of growing tumor in mice.

We established a mouse rising dbl quote, left (low)primary tumor resection model" in which a transplanted tumor was resected after an orthotopic transplantation of colorectal cancer tissue to estimate the therapeutic effect of an angiogenesis inhibitor on metastasis. The angiogenesis inhibitor FR-118487 is a member of the fumagillin family. Here, 1 mg / kg / day of FR-118487 was subcutaneously administered to nude mice for 1 week, 2 weeks, or 4 weeks through an osmotic pump. Liver metastasis developed in 7 of 9 control mice, 2 of 6 mice that underwent the tumor resection 2 weeks after transplantation (early resection), and in all 7 of the mice that underwent the tumor resection 4 weeks after transplantation (late resection). In the short treatment trial, the FR-118487 administration immediately after the early resection completely inhibited both hepatic and peritoneal metastases, whereas its administration after the late resection had no effect on liver metastasis. In the prolonged treatment trial, inhibitory effects of prolonged treatment with FR-118487 on both hepatic and peritoneal metastases after the late resection were clearly demonstrated. The mice of the resection-alone group all died within 106 days after tumor inoculation, due to metastases of colon carcinoma. In contrast, half of the mice that underwent resection and then received antiangiogenic therapy were alive at the end of the observation period (160 days after transplantation). In conclusion, the combination of surgery and subsequent antiangiogenic therapy may be useful to prevent the distant metastasis of colorectal cancer and to improve the prognosis of patients with colorectal cancer.     

Marked granulocytosis in C57BL/6 mice bearing a transplanted BMT-11 fibrosarcoma

When a 3-methylcholanthrene (CAS: 56-49-5)-induced fibrosarcoma, BMT-11, and its eight clones were transplanted sc into syngeneic C57BL/6 mice, leukemoid reaction characterized by a progressive increase in peripheral white blood cells (WBCs) and by splenomegaly was observed as the tumors grew. The WBC count reached about forty-fold of the normal level, and more than 90% of WBCs were found to be polymorphonuclear leukocytes. The increase in WBCs was correlated with tumor size, and its count decreased to normal level within 7 days after surgical excision of subcutaneous tumors. Moreover, a high level of colony-stimulating activity was detected in the supernatant of BMT-11 culture. These results suggest that the colony-stimulating factor produced by BMT-11 cells caused granulocytosis in mice. This is the first report of the marked degree of granulocytosis induced by a transplanted tumor in C57BL/6 strain mice.     

In Vivo Confocal Fluorescence Imaging of the Intratumor Distribution of the Photosensitizer Mono-l-Aspartylchlorin-e6

We present an in vivo fluorescence microscopic evaluation of intratumor distribution of the photosensitizer mono-l-aspartylchlorin-e6 (NPe6) in an intradermal mouse EMT6 tumor model. Although the identification of favorable photophysical and pharmacological properties has led to the development of new photosensitizers in photodynamic therapy, their intratumor distribution kinetics have remained relatively understudied. In this study, we used confocal fluorescence microscopy to follow the transport of NPe6 in vivo after systemic administration through the tail vein. Labeling of vasculature using fluorophore-conjugated anti-CD31 antibodies allows visualization of the uptake of NPe6 in tumor and normal vessels and its partitioning kinetics into the adjacent parenchyma for 3 hours after injection. During the initial 60 minutes after injection, the drug is predominantly confined to the vasculature. Subsequently, it significantly redistributes throughout the extravascular regions with no discernable difference in its extravasation rate between tumor and normal tissues. Further, we investigate the sensitizer''s altered intratumor distribution in response to photodynamic therapy irradiation and observe that treatment-induced changes in vessel permeability caused enhanced accumulation of NPe6 in the extravascular space. Our findings are of immediate clinical relevance and demonstrate the importance of an in vivo imaging approach to examine the dynamic process of intratumor drug distribution.     

羟基红花黄色素A对小鼠Lewis肺癌移植瘤的抑制作用

目的 研究羟基红花黄色素A(hydroxysafflor yellow A,HSYA)对C57BL/6小鼠Lewis肺癌移植瘤的血管抑制作用.方法 C57BL/6近交系小鼠30只,造模后,分为5组,每组6只,分别使用生理盐水、环磷酰胺(CTX)及HSYA(分低、中、高3个剂量)作用于动物模型.观察各组肿瘤体积大小,分析小鼠生长曲线,并在造模第22天处死所有小鼠,无菌条件下剥取瘤组织进行HE染色,观察不同浓度HSYA在不同视野下对肿瘤血管的抑制情况.结果 HSYA中、小剂量组对小鼠移植瘤血管生成有一定抑制作用.结论 中药羟基红花黄色素A(HSYA)对Lewis肺癌小鼠移植瘤血管生成有一定的抑制作用.     

Hair follicle-derived blood vessels vascularize tumors in skin and are inhibited by Doxorubicin

We have recently shown that the neural-stem cell marker nestin is expressed in hair follicle stem cells and the blood vessel network interconnecting hair follicles in the skin of transgenic mice with nestin regulatory element-driven green fluorescent protein (ND-GFP). The hair follicles were shown to give rise to the nestin-expressing blood vessels in the skin. In the present study, we visualized tumor angiogenesis by dual-color fluorescence imaging in ND-GFP transgenic mice after transplantation of the murine melanoma cell line B16F10 expressing red fluorescent protein. ND-GFP was highly expressed in proliferating endothelial cells and nascent blood vessels in the growing tumor. Results of immunohistochemical staining showed that the blood vessel-specific antigen CD31 was expressed in ND-GFP-expressing nascent blood vessels. ND-GFP expression was diminished in the vessels with increased blood flow. Progressive angiogenesis during tumor growth was readily visualized during tumor growth by GFP expression. Doxorubicin inhibited the nascent tumor angiogenesis as well as tumor growth in the ND-GFP mice transplanted with B16F10-RFP. This model is useful for direct visualization of tumor angiogenesis and evaluation of angiogenic inhibitors.     

510.6nm绿色铜蒸气激光加血卟啉衍生物(HpD)对动物移植瘤杀伤作用的实验研究

本文报告510.6nm绿色铜蒸气激光与HpD对动物移植瘤的杀伤作用.给带S_(180)和B_(16)—MB小鼠腹腔注射HpD50mg/kg,注后72小时,用510.6mm铜蒸气激光照射肿瘤,照后肿瘤体积和重量,均明显减少(P<0.01和<0.05),病理检查照区肿瘤均有坏死,深度为5.5～8.8mm,平均约7mm,单纯铜激光照射,肿瘤坏死深度最大达5mm.HpD组和非激光非HpD组无上述变化.HpD对小鼠B_(16)—MB生长抑制试验,未显示其本身抑制肿瘤生长的作用.本实验结果表明510.6nm铜激光对癌细胞杀伤作用是肯定的,可作为浅表癌肿PDT治疗的光源.     

Photodynamic therapy for lung cancers based on novel photodynamic diagnosis using talaporfin sodium (NPe6) and autofluorescence bronchoscopy

BACKGROUND: We had previously developed the possibility of use of a photodynamic diagnosis (PDD) system using a tumor-selective photosensitizer and laser irradiation for the early detection and photodynamic therapy (PDT) for centrally located early lung cancers. Recently, we established the autofluorescence diagnosis system integrated into a videoendoscope (SAFE-3000) as a very useful technique for the early diagnosis of lung cancer. PATIENTS AND METHODS: Twenty-nine patients (38 lesions) with centrally located early lung cancer received PDD and PDT using the second-generation photosensitizer, talaporfin sodium (NPe6). Just before the PDT, we defined the tumor margin accurately using the novel PDD system SAFE-3000 with NPe6 and a diode laser (408nm). RESULTS: Red fluorescence emitted from the tumor by excitation of the photosensitizer by the diode laser (408nm) from SAFE-3000 allowed accurate determination of the tumor margin just before the PDT. The complete remission (CR) rate following NPe6-PDT in the cases with early lung cancer was 92.1% (35/38 lesions). We also confirmed the loss of red fluorescence from the tumors immediately after the PDT using SAFE-3000. We confirmed that all the NPe6 in the tumor had been excited and photobleached by the laser irradiation (664nm) and that no additional laser irradiation was needed for curative treatment. CONCLUSIONS: This novel PDD system using SAFE-3000 and NPe6 improved the quality and efficacy of PDT and avoided misjudgement of the dose of the photosensitizer or laser irradiation in PDT. PDT using NPe6 will become a standard option of treatments for centrally located early lung cancer.     

肿瘤组织骨髓来源新生血管内皮和炎症细胞浸润动物模型的观察

背景与目的:肿瘤组织新生血管主要是靠宿主血管以出芽的方式形成,一些研究认为骨髓来源的内皮前体细胞(endothelial progenitor cells,EPCs)参与肿瘤组织血管的生成,而且肿瘤组织中骨髓来源的炎症细胞可能促进肿瘤的侵袭、血管生成和转移等。本实验旨在建立动物模型以观察骨髓来源的肿瘤组织新生血管内皮细胞,以及骨髓来源的炎症细胞在瘤组织中的浸润情况。方法:以绿色荧光蛋白(green fluorescent protein,GFP)转基因C57BL/6小鼠为供体提供骨髓,普通清洁级C57BL/6小鼠经60Co 8 Gy全身照射,24h后为受体进行骨髓移植,于移植2周后接种Lewis肺肿瘤细胞于受体小鼠腋下,待肿瘤直径达到1～2cm时,取肿瘤组织切片在荧光显微镜下直接观察肿瘤血管及炎症细胞,并结合免疫组化方法明确其种类和分布。结果:在荧光显微镜下可观察到散发不连续性绿色荧光的肿瘤血管内皮细胞和浸润的炎症细胞,经免疫组化测定染色大部分呈阳性。肿瘤细胞周边和内部坏死区可见大量巨噬细胞浸润;肿瘤间质和肿瘤细胞区散在分布着少量淋巴细胞。结论:肿瘤组织新生血管的部分内皮细胞来源于骨髓;这种动物模型可以作为观察肿瘤组织中骨髓来源细胞特异而直接的方法。