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
2、。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 ca
3、pacity, and every fragment is capable of growing into a complete 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 me
4、dium volumes, and can be kept inexpensively in laboratory 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 surfac
5、tants on oxidative stress and cholinesterase(ChE) activities 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 con
6、centrations of 40, 80, and 160 gL1, suggesting that 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,
7、 behavior, and morphology in each species5. In addition, 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 re
8、quired for autophagy can function at the interface 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 reg
9、eneration of planarians was significantly retarded 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
10、addition, the putrescine and spermidine levels could 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,
11、are involved in multiple biological processes in organisms, 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. Moreov
12、er, the possible associations of development with lipid 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 maintaine
13、d in autoclaved water at 22 , and fed with chicken 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 assa
14、y of development and accumulation of lipid and glycogen.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 pla
15、narians was examined to represent the body length. 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
16、frozen sections were cut at 812 m using freezing microtome(Leica CM1900, Germany). The sections were stained with 10 mgml-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 exp
17、eriments were repeated at least three times.1.5 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 microtom
18、e(Leica CM1900, Germany). Sections were treated 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. C
19、ontrol sections were pretreated with aamylase. 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 genera
20、ted using Microsoft Excel(Microsoft Corp., 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 o
21、f body weight with accumulation of lipid 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 st
22、udy, the development of planarians was 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 tre
23、ated planarians were all significantly(P0.01) decreased compared with those in control planarians without heatshock treatment.Bars represent means S.D. Control, without heatshock treatment. a. P0.01 vs controlFig 1 Effects of heatshock on body weight of planarians2.2 Effects of heatshock on body len
24、gth of planarians We next 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, P0.05; 20, 24, 28, and 32 h, P0.01) decreased compared
25、with those in control 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. P0.05 vs control, b. P0.01 vs controlFig 2 Effects of heatshock on b
26、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
27、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(P0.01) reduced compared with those in control plana
28、rians without heatshock treatment(Fig 3B). Therefore, treatment with the stress of heatshock will inhibit the lipid accumulation in parenchymal cells of planarians.2.4 Effects heatshock on glycogen accumulation in planarians Considering the fact that glycogen is another important carbohydrate in liv
29、ing organisms, we further investigated the effects of heatshock treatment on glycogen accumulation in planarians. As shown in Fig 4, after heatshock treatment for different time intervals, the relative intensities of labeled signals for glycogen in parenchymal cells were significantly(P0.01) decreas
30、ed compared with those in control planarians without heatshock treatment. Therefore, treatment with the stress of heatshock will also suppress the glycogen accumulation in parenchymal cells of planarians.A. The pictures of lipid accumulation in parenchymal cells of control and 24 h heatshock treated
31、 planarians. B. Comparison of the relative sizes of lipid droplets in parenchymal cells between control and heatshock treated planarians. Control, without heatshock treatment. a. P0.01 vs controlFig 3 Effects of heatshock on lipid accumulation in planarians 2.5 Associations of development with the a
32、ccumulation of lipid or glycogen in heatshock treated planarians Linear regression analysis further suggests that the body weight was significantly(P0.01) correlated with the relative size of lipid droplet(R2=0.787), and the relative intensity of glycogen signal(R2=0.814) in heatshock treated planar
33、ians(Tab 1). Similarly, the body length was significantly(P0.01) correlated with the relative size of lipid droplet(R2=0.801), and the relative intensity of glycogen signal(R2=0.884) in heatshock treated planarians(Tab 2). Therefore, the close association between the development and the accumulation
34、 of lipid or glycogen exists in heatshock treated planarians.A.The pictures of glycogen accumulation in parenchymal cells of control and 24 h heatshock treated planarians. B. Comparison of the relative intensities of labeled signals for glycogen in parenchymal cells between control and heatshock tre
35、ated planarians. Control, without heatshock treatment. a. P0.01 vs controlFig 4 Effects heatshock on glycogen accumulation in planarians Tab 1 Associations of body weight with the accumulation of lipid or glycogen in heatshock treated planarians as assayed by linear regression analysisDependentIndep
36、endentPartial R2Pvariablevariablebody weightRelative size of lipid droplet0.7870.01Relative intensity of glycogen signal0.8140.01Tab 2 Associations of body length with the accumulation of lipid or glycogen in heatshock treated planarians as assayed by linear regression analysisDependentIndependentPa
37、rtial R2Pvariablevariablebody weightRelative size of lipid droplet0.8010.01Relative intensity of glycogen signal0.8840.013 DiscussionPrevious studies have shown that several kinds of stresses can induce the adverse alterations of development and reproduction in planarians3-7. Our data in the current work further suggest that exposure to heatshock can also cause the defects of development in planarians. Heatshock treatment for different time intervals caused the significant decrease of body weight compared with those in control planarians(Fig 1).
