热激胁迫可能通过降低脂肪与糖原的积累抑制涡虫的发育

1、热激胁迫可能通过降低脂肪与糖原的积累抑制涡虫的发育         11-03-25 13:23:00     编辑：studa20                       作者：刘培党,林兴凤,王大勇 【摘要】  目的:探讨热激胁迫对于涡虫发育

2、以及脂肪和糖原积累的影响。方法:涡虫的发育通过体长与体质量来评价。分析35 热激胁迫处理涡虫的体长与体质量变化以及实质细胞中脂肪与糖原积累情况,并评价热激胁迫处理涡虫中发育与脂肪或糖原积累之间可能的相关性。结果:与未经历热激处理的对照相比,热激胁迫处理不同长度时间(16、20、24、28与32 h)后涡虫体长与体质量显著减少或降低。与对照相比,热激胁迫处理导致涡虫实质细胞中的脂肪颗粒数量明显降低、尺寸显著变小。类似地,热激胁迫处理还导致涡虫实质细胞中的糖原积累的显著降低。进而,线性回归分析表明热激胁迫处理涡虫的体长与体质量均显著相关于实质细胞中脂肪颗粒的尺寸或糖原的含量。结论:热激胁迫处理可以

3、明显抑制涡虫的发育,且热激胁迫处理涡虫中存在发育与脂肪或糖原积累之间的显著关联。 【关键词】  热激胁迫; 脂肪; 糖原; 发育; 涡虫Freshwater freeliving planarians, an important component of the aquatic ecosystem, are distributed worldwide in unpolluted streams. Planarians have a high regenerative capacity, and every fragment is capable of growing into a c

4、omplete individual if an individual planarian is cut into small fragments. Traditionally, planarians have been a favored model animal in developmental biology. They can be easily collected in large numbers, require only low culture and test medium volumes, and can be kept inexpensively in laboratory

5、 for the study of stem cell biology1, neuroregeneration2, and toxicology3.So far, the characteristics of planarian make it a suitable organism for studying the effects of environmental stresses. The acute toxicities of eight widely used surfactants on oxidative stress and cholinesterase(ChE) activit

6、ies were examined in planarian Dugesia japonica, and the differences in acute toxicity among these surfactants were at least in the range of three orders of magnitudes3. The catalase activity was significantly induced by copper exposure at concentrations of 40, 80, and 160 g·L1, suggesting that

7、 the catalase levels in planarians could represent a biomarker for estimation of copper effects in freshwater ecosystem4. The effects of UVirradiation to two planarian species were also determined, and UVrays caused obvious alterations of mortality, behavior, and morphology in each species5. In addi

8、tion, the number of neoblasts extremely increased in the area of damaged tissue, immediately after UV irradiation5. Hypertonic osmotic shock treatment could induce the temporarily increase of putrescine and spermidine levels6. Moreover, the genes required for autophagy can function at the interface

9、between survival and cell death during stressinducing processes like regeneration and starvation in sexual and asexual races of planarians7.The effects of low temperature on reproduction and regeneration have been investigated in planarians. The regeneration of planarians was significantly retarded

10、for all levels by lowering the temperature8. Vowinckel(1970) showed that exposure to low water temperatures stimulated the increase in germ cells associated in testicular primordial and that this activity could be transmittable via homogenates9. In addition, the putrescine and spermidine levels coul

11、d be temporarily increased after coldshock treatment in planarians6. Nevertheless, the possible effects of heatshock on the planarian development are still largely unclear.Considering the fact that complex carbohydrates, such as lipid and glycogen, are involved in multiple biological processes in or

12、ganisms, the first objective of this study was to investigate the effects of heatshock on development of planarians, and the second objective of this study was investigate the alterations of lipid and glycogen in heatshock treated planarians. Moreover, the possible associations of development with l

13、ipid or glycogen storage were examined in heatshock treated planarians.1 Materials and methods1.1 AnimalsThe planarians(Dugesia japonica) were collected from the stream in the Zijin Mountain, Nanjing, in 2009. The planarians were asexually maintained in autoclaved water at 22 , and fed with chicken

14、liver twice a week. Planarians were starved for more than 1 week prior to the experiments. The planarians were treated with heatshock at 35 for 16, 20, 24, 28, and 32 h. The planarians with the approximately same size(5 mm length) were used for assay of development and accumulation of lipid and glyc

15、ogen.1.2 Body weightTo calculate the average body weight of planarians, 10 animals were weighed after the water on the surface of animals was cleaned with filter paper. The experiments were repeated six times.1.3 Body lengthThe midline length of planarians was examined to represent the body length.

16、The experiments were repeated six times.1.4 Lipid stainingA modified sudan black B staining method was used to evaluate the lipid accumulation in parenchymal cells as previously described10. Planarians were fixed in Bouins solution for 8 h, and the frozen sections were cut at 812 m using freezing mi

17、crotome(Leica CM1900, Germany). The sections were stained with 10 mg·ml-1 sudan black B solution, and the stained lipid droplets were dark blue. The diameters of stained lipid droplets were calculated to reflect the size of lipid droplet, and the experiments were repeated at least three times.1

18、.5 Glycogen stainingThe periodic acidschiff(PAS) method for glycogen staining in parenchymal cells was performed as previously described11. Planarians were fixed in Bouins solution for 8 h, and the frozen sections were cut at 812 m using freezing microtome(Leica CM1900, Germany). Sections were treat

19、ed with 0.5% periodic acid for 10 min, rinsed in water for 10 min, and stained with schiff reagent for 10 min. Moreover, the sections were rinsed with sodium pyrosulfite for 2 min, washed in water for 5 min, and dehydrated and mounted in neutral balsam. Control sections were pretreated with aamylase

20、. The stained glycogen was red color in the cytosol. The intensities of stained glycogen were examined, and the experiments were repeated at least three times.1.6 Statistical analysisAll data in this article were expressed as means ± S.D. Graphs were generated using Microsoft Excel(Microsoft Co

21、rp., Redmond, WA). Oneway analysis of variance(ANOVA) followed by a Dunnetts ttest was used to determine the significance of the differences between the groups. The probability levels of 0.05 and 0.01 were considered statistically significant. The associations of body weight with accumulation of lip

22、id or glycogen, as well as the associations of body length with accumulation of lipid or glycogen, were examined with the method of linear regression in heatshock treated planarians.2 Results2.1 Effects of heatshock on body weight of planarians In the present study, the development of planarians was

23、 evaluated by the endpoints of body weight and body length. We first investigated the effects of heatshock on body weight. As shown in Fig 1, after heatshock(35 ) treatment for different time intervals(16, 20, 24, 28, and 32 h), the body weights of heatshock treated planarians were all significantly

24、(P<0.01) decreased compared with those in control planarians without heatshock treatment.Bars represent means ± S.D. Control, without heatshock treatment. a. P<0.01 vs controlFig 1 Effects of heatshock on body weight of planarians2.2 Effects of heatshock on body length of planarians We ne

25、xt examined the effects of heatshock on body length. As shown in Fig 2, similarly, after heatshock treatment for different time intervals, the body lengths of heatshock treated planarians were also all significantly(16 h, P<0.05; 20, 24, 28, and 32 h, P<0.01) decreased compared with those in c

26、ontrol planarians without heatshock treatment. Therefore, treatment with the stress of heatshock can obviously suppress the development of planarians.Bars represent means ± S.D. Control, without heatshock treatment. a. P<0.05 vs control, b. P<0.01 vs controlFig 2 Effects of heatshock on b

27、ody length of planarians2.3 Effects of heatshock on lipid accumulation in planarians Again, we examined the effects of heatshock treatment on lipid accumulation in planarians. As shown in Fig 3A, compared with the lipid accumulation in control planarians without heatshock, heatshock treatment result

28、ed in the sharp decrease of number of lipid droplets in parenchymal cells. Moreover, after heatshock treatment for different time intervals, the relative size of lipid droplets in parenchymal cells of heatshock treated planarians were significantly(P<0.01) reduced compared with those in control planarians without heatshock treatment(Fig 3B). Therefore, treatment with the stress of heatshock will inhibit the lipid accumulation in parenchymal cells of