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1、 Actinomycin D VM-26 on rat glioma cell proliferation in vivo study of the effects ofAuthors: WU Yong-Kang, Lun, Hua-Wei Liu, Shen Lin Abstract Objective To study the actinomycin D (Actinomycin D, ACTD), and teniposide (Teniposide, VM-26) on rat glioma cells in vivo therapeutic effect. Methods of wi
2、ld-type C6 rat glioma cells inoculated in mouse brain on the right caudate nucleus (control group 10, no-load group of 10 rats), and has been the formation of C6 rat brain glioma and VM respectively ACTD -26 anti-tumor in situ injection of sustained-release agent tumor area (treatment group = 10). O
3、bserved in rats in each group in general, survival, tumor pathology and magnetic resonance imaging (MRI) dynamic change, using PCNA counts, TUNEL assay of tumor cell proliferation activity and apoptosis. Results In the control group and no-load average survival period of rats 19.3 days, ACTD Section
4、 6, and VM-26 group 3 rats were significantly prolonged survival time, survival period of more than 200 days; In addition to human death due to pathological examination of two things , ACTD group died within 4 weeks after treatment 2, VM-26 Section 5. MRI examination in control group significantly i
5、n rat brain tumor foci, the treatment group in the rat brain tumor foci decreased or disappeared after treatment, are consistent with the pathological examination, and the treated rats reduced C6 cell proliferation, a large number of cells, wither death, ACTD significantly higher than VM-26. Conclus
6、ion ACTD local application in vivo is expected to become the preferred treatment of glioma chemotherapy. Keywords: actinomycin D; Teniposide; nerve glioma; body; chemotherapy Abstract Objective To study the therapeutic effect of ACTD and VM-26 on rat C6 glioma in vivo.Methods Wild type C6 cells were
7、 implanted stereotaxically to the caudate nucleus of right cerebrum of Wistar rats (control and empty loading group, 10 rats respectively ), and rats with cerebral tumor foci were treated ACTD and VM-26 mediated by delayed release retarder (treated group, 10 rats respectively). The general manifesta
8、tion, survival time, MRI features, histopathological change, proliferation activity and apoptosis of the glioma in each group of rats were observed.Results The mean survival time of control animals was 19.3 days.Two ACTD or VM-26 treated rats were killed on day 28 and 58 after implantation for histo
9、pathological examination.The surviving time of the 9 remaining rats (6 ACTD and 3 VM-26 treated rats) was over 200 days.MRI demonstrated a distinct cerebral tumor in control rats, but the tumor foci were disappeared almost completely in the treated rats.The cerebral glioma of rats after the treatmen
10、t with ACTD showed decrease in proliferation activity and apoptosis, significantly decrease than the treatment with VM-26.Conclusion The results of ACTD in treatment of rat C6 glioma is encouraging.It can be used as the chemotherapy for human malignant glioma. Keywords: ACTD; VM-26; glioma; in vivo;
11、 chemotherapy Our previous results show that actinomycin D (Actinomycin D, ACTD) can significantly inhibit the proliferation of C6 rat malignant glioma cells induced apoptosis, in contrast to four different concentrations of the anti-tumor effect in the ACTD are stronger than the Teniposide (Tenipos
12、ide, VM-26), apoptosis rate in the high-dose ACTD was superior when the VM-26 1. To further clarify the efficacy of both chemotherapy of malignant glioma, we studied the C6 cells were tumorigenic mouse brain, as well as rat brain has been formed C6 glioma tumor foci application of ACTD and VM-26 ant
13、i-tumor slow release agent tumor efficacy of the treatment zone in situ were observed in rats in general, the survival period, tumor volume change and tumor histopathological characteristics and changes in cell biology. 1 Materials and methods 1.1 The experimental material C6 rat glioma cells in ani
14、mals provided by the Chinese Academy of Sciences. Cell culture medium: DMEM (Dulbecco modified eagle medium Gibco, USA), fetal calf serum (Hyclone, USA). Main biochemical reagent: The disinfection of medical gelatin 10g adding water for injection 50ml, was added ACTD 0.1g and VM-26 0.5mg, stirred in
15、to a semi-liquid-like, made of slow-release anti-tumor agent. In Situ Apoptosis Detection Kit (Roche, Germany), ABC immunohistochemistry kits and antibody (Beijing Zhongshan and their agents Santa Cruz companies). Main instruments: jiangwan type stereotactic frame (Second Military Medical University
16、 of production), magnetic resonance instrument (Advantage 1.5 Tesla GE Medical Systems, GE Corporation, USA). Experimental animals: two male SD rats weighing 200 300g, a total of 40, from Beijing and biological products propagation provide on the spot, its characteristics have been identified. 1.2 E
17、xperimental Methods 1.2.1 C6 rat glioma model 1 C6 rat glioma cells containing 10% fetal calf serum DMEM medium monolayer culture. Inoculation cell volume of 1.0 * 106 / (10 l * only). Rats by 10% chloral hydrate (300mg/kg) by intraperitoneal injection anesthesia, fixed in Riverside type stereotacti
18、c frame, the surgical exposure to the skull logo, the former top of head position, the right side of sagittal suture at 3.0 3.5mm with the dental drill drilling, aperture 1.2mm, with micro-syringe needle perpendicular to the right caudate nucleus, the cell suspension slowly injected. 1.2.2 Experimen
19、tal animal groups and brain tissue samples collected were divided into four experimental exercise groups, each of 10 SD rats were: (1) control group: inoculation of wild-type C6 rat glioma cells; (2) No-load slow Release Agent Group: vaccination of wild-type C6 rat glioma cells after tumor multi-poi
20、nt injection of semi-liquid-like medical gelatin; (3) ACTD treatment groups: treatment group, C6 rat glioma cells 5 days after implantation, MRI confirmed tumor formation, respectively, in the first days by the above method 6,28 anesthesia, fixed animals; the same time, sustained-release preparation
21、 of a good ACTD anti-tumor agent in-situ multi-point injection of the tumor; (4) VM-26 treatment groups: treatment group C6 rat glioma cells 5 days after implantation, MRI confirmed tumor formation, respectively, in the first six days according to the above method of anesthesia, fixed animals; the s
22、ame time, a good VM-26 Preparation of sustained-release anti-tumor agent in-situ multi-point injection into the carrier tumor. 28,58 days after inoculation were sacrificed 1, tumor specimens were conventional HE staining and detection of apoptosis; the remaining eight long-term observation of surviv
23、al. Experimental animals in each group died of natural causes or man-made in their brain tissue removed after death, the tumor samples in 10% formalin fixed, paraffin-embedded after routine coronal slices for gross specimen observation, routine HE staining; the other to PCNA immunohistochemical stai
24、ning. Another part of the specimen with liquid nitrogen quick-frozen OCT-embedded frozen sections stored -70 , line in situ cell apoptosis detection. 1.2.3 MRI imaging and detection methods 2 with the machine, GE 1.5T Signa MR imaging, coronal and sagittal T1WI plain and enhanced scanning. Based on
25、animal model control group, the law of survival in this experimental treatment group and no-load group of 10 rats in 5 days after planting for all the samples to understand the situation into a tumor to treatment. In rats 1 week after planting four animals were randomly selected for testing. After t
26、he continuous detection of tumor growth in rats in each group changes in the specific time of 2 weeks, 4 weeks, 8 weeks, 12 weeks. Each test, in addition to select at least three growing after 5 days or 1 week have been carried out continuous monitoring of test animals, other animals, then each choo
27、se a 2 rotation detection, understanding of the animals into the tumor rate. Tumor volume calculation: In the experimental design requirements for point in time, under the enhanced scan image in the coronal plane measurement of the largest tumor maximum tumor diameter length (L) and wide-diameter (W
28、), the largest tumor in the sagittal plane scan measurement of height to diameter (H), substituted into the formula: V = (4 / 3 * * L * W * H) * 1 / 8. 1.2.4 Tissue specimens of conventional HE staining and apoptosis detection see reference 1 3, methods omitted. 2 Results 2.1 The general situation a
29、nd survival in rats observed in control group and no-load Group: C6 cells 1 week after inoculation, rats were significantly reduced foraging activity of drinking water, weight loss, was followed by progressive left hemiplegia, and are at 2 to 3 weeks die within. 8 The average survival period of rats
30、 (19.3 4.9) days. 2.2 The treatment group was the first 2 3 weeks rats were generally similar to the control group, in a dying state. ACTD group of 10 rats were inoculated, respectively, in the first three days 16,21,37 death; the other six rats did not die from the first four weeks of drinking wate
31、r from feeding activities of gradually returning to normal, weight gain, of which two, respectively, 28,58 days after inoculation executed three death row in the first 200 days, pathological examination. VM-26 treatment group, 10 rats were inoculated, respectively, in 5 days after the death 16,18,22
32、,36,41; the other five are not dead rat from the first four weeks of drinking water from feeding activities of the gradual return to normal, weight gain, of which two, respectively, 28,58 days after inoculation executed three death row the first 200 days, pathological examination. Reposted elsewhere
33、 in the paper for free download 2.3 MRI imaging and the corresponding pathological changes in rats on the 4 regular inspections MRI, and was killed random inspection to observe the pathological changes accordingly. 2.3.1 inoculated C6 cells in the control group 1 week after tumor area showed a long
34、T1 long T2 signal light, mild homogeneous or inhomogeneous enhancement with mild midline shift, tumor size, respectively 155,150,186,172 mm3. 2 weeks after the tumor foci for long T1 long T2 signal, uniform enhancement or ring-like enhancement, midline shift evident, in which three ventricles of the
35、 brain expanded, tumor lesion volume 410,740,546,630,590 mm3. Pathological examination showed the growth of C6 cells active, there are cystic tumor, necrosis, Ce Shi choroid plexus, subependymal and subarachnoid seen a small amount of tumor cells. Empty set of results similar to the control group. 2
36、.3.2 ACTD treatment group 1 week after inoculation and 2 weeks the results observed with the control group. 4 weeks after inoculation and 8 weeks of the original found to enhance tumor foci gradually reduced, midline mild left ventricular expansion. Vaccination at 12 weeks and 28 weeks of review, tu
37、mor foci disappeared, no displacement of midline structures. The first 200 days in both the pathological examination of brain substance no tumor cells, Ce Shi choroid plexus, subependymal and subarachnoid seen a small amount of non-proliferating C6 cells (see Figure 1). A: After 1 week of tumor inoc
38、ulation; B: After 2 weeks of tumor inoculation; C: After 4 weeks of tumor inoculation; D: inoculation into the tumor 12 weeks after 2.3.3 VM-26 treatment group, treatment group, similar to results with ACTD, but the extent of tumor shrink tumor foci than ACTD group. And 28 weeks when the two are sti
39、ll remnants of tumor foci. 2.4 Determination of tumor cell proliferation activity in the control group and the average no-load group of tumor cell PCNA count 8.92 5.29. ACTD-treated group, three in inoculation 2 3 weeks after the death, the count of PCNA in tumor tissue were 4.05,3.91,5.45; and 28,5
40、8 days after inoculation at the dead of their tumor tissue PCNA counts were 2.45 and 1.83. VM-26 treatment group, five in the inoculated 2 3 weeks after the death, the count of PCNA in tumor tissue were 5.05,3.21,6.20; and 28,58 days after inoculation at the dead of their tumor were PCNA count 3.55
41、and 3.42. 2.5 detection of apoptosis in control group and ACTD and VM-26 treatment group died in the treatment fails C6 tumor-bearing mice occasionally apoptosis of tumor cells; no-load group can see that a small amount of apoptosis of tumor cells; while ACTD and VM-26 treatment group in the first 2
42、8 days after inoculation and were sacrificed 58 days in rat C6 tumors can be seen a large number of apoptotic cells, the former should be greater than the latter. 3 Discussion In the rat brain glioma cell line inoculated with the kinds of established glioma animal model to test an important means of
43、 various treatment methods 2 4. We use MR imaging dynamic observation of C6 rat glioma model and the changes after treatment, easy to observe the intracranial glioma growth and the changes in dynamic observation of tumor foci to help explore its growth pattern and changes after treatment 4,5 . VM-26
44、 for a table of artificial semi-synthetic derivatives of podophyllotoxin for the cycle specific cytotoxic drugs, acting on the late S2 and G2 cell cycle phase, by preventing cells to enter mitotic phase and play a role, but also by inhibiting DNA topoisomerase cause DNA single-strand and double-stra
45、nd breaks, resulting in cytotoxicity. Its molecular weight is small, fat-soluble high, through the blood-brain barrier easily applied to the treatment of brain tumors, apoptosis. However, in vivo experiments show that efficient use of drugs alone is not high, and increase the dose can cause serious
46、side effects, mainly bone marrow suppression, mainly for the white blood cells, thrombocytopenia. The ACTD is a phthalic ester type anti-tumor antibiotics, anti-tumor mechanisms and VM-26 is different from the glioma cells through activation of cysteine peptidase -3 and effective induction of apopto
47、sis play a role in cell killing, commonly used in clinical the treatment of malignant mole, uterine choriocarcinoma, melanoma, etc., the exact efficacy, toxicity is relatively light. Because of its easy through the blood-brain barrier, which previously seldom used in the treatment of intracranial tu
48、mors. Recently, we have in the C6 glioma cells in vitro experiments found that in the comparison of different molar concentration gradient, at all levels of value-added of ACTD inhibit tumor was significantly superior to VM-26 1. Naritan such as the recent in vitro tumor inhibition test also proved
49、its glioblastoma tumors with a very strong inhibition 6. ACTD and the application of slow-release agent to the tumor can be localized directly on tumor cells, to maximize its excellent anti-tumor effect and enhance the local concentration of the drug and reduce toxicity and overcome the blood-brain barrier blocking effect. Our test results showed that, ACTD treatment group 1 week after