» arhiv
Možnosti:
[Prikaži v obliki za tiskanje]
[Poišči nalogo v zbirki Cobiss]
ID naloge: 4 Letnik: 1997 Predmet: patofiziologija
Razporeditev jeder in pripadajoce celokupne rna
v normalnem in denerviranem mišicnem vlaknu
Avtor: Tanja Milanic, Andrej Kunstelj
Mentor: Prof. dr. Zoran Grubic
IZHODIŠCA: Za razliko od vecine tkiv v sesalskem organizmu, kjer je osnovni gradbeni element enojedrna celica, je skeletnomišicno vlakno vecjedrni sincicij. Takšna organizacija tkiva omogoca regulacijo beljakovinske sinteze na ravni razporeditve in specializacije mišicnih jeder, saj gre pri razporejanju jeder vzdolž mišicnega vlakna pravzaprav za razporejanje genov. Vecja ali manjša koncentracija genov lahko pomembno vpliva na koncentracijo transkriptov, in s tem na hitrost sinteze beljakovin.
NAMEN: V okviru naše naloge smo se namenili ugotoviti, v kolikšni meri morfometricne znacilnosti razporeditve jeder in njihove oblike v normalnem in denerviranem mišicnem vlaknu podpirajo opisano razmišljanje. Odlocili smo se preveriti naslednje hipoteze:
1. Gostota jeder v podrocju motoricne plošcice presega gostoto v zunajplošcicnem podrocju hitre mišice za faktor, vecji od 10; pri takšnem faktorju bi že lahko pricakovali pomembne razlike tudi v koncentraciji genskih produktov med plošcicnim in zunajplošcicnim podrocjem.
2. Velikost plošcicnih jeder se statisticno pomembno razlikuje od velikosti zunajplošcicnih
jeder.
3. Za organizacijo plošcicnega jedrnega skupka so pomembni živcni dejavniki, zato bo po denervaciji prišlo do znižanja gostote jeder v plošcicnem podrocju in do zmanjšanja morfoloških razlik med plošcicnimi in zunajplošcicnimi jedri.
4. Po denervaciji se množina celokupne RNA, ki pripada posameznemu jedru, ne spremeni, kar pomeni, da lahko porast celokupne RNA v mišici v teh razmerah pripišemo zgošcevanju že obstojecih jeder in njihovemu pomnoževanju na racun delitve satelitnih celic.
METODE: Razvili smo metodo, ki temelji na izolaciji posameznih mišicnih vlaken ter na istocasnem obarvanju mišicnih jeder (s fluorescencnim Hoechstovim barvilom) in motoricnih plošcic (s tioholinsko metodo) v teh vlaknih. Morfometricne parametre smo dolocali s pomocjo analizatorja slike. Celokupno RNA smo izolirali po gvanidinium-tiocianatni metodi. Poskuse smo opravljali na hitrih vlaknih podganje mišice m. sternomastoideus. Pri statisticnih analizah smo uporabljali Studentov neparni t-test.
REZULTATI: Koncentracija jeder v plošcicnem podrocju za faktor 17 presega koncentracijo jeder izven tega podrocja. Najdaljša os elipticnih plošcicnih jeder je statisticno znacilno (p<0.05) krajša od iste osi zunajplošcicnih jeder, po najkrajši osi pa se jedra obeh podrocij ne razlikujejo (p>0.05). Tako sedem kot štirinajst dni po denervaciji nismo ugotovili znacilnih sprememb v številu in velikosti plošcicnih jeder, statisticno znacilno (p<0.05) pa se je zmanjšala dolžina osi zunajplošcicnih jeder. Po sedemdnevni denervaciji smo ugotovili neznacilno (p>0.05) povecanje celokupne RNA v celi mišici in statisticno znacilno (p<0.05) povecano množino RNA na utežno enoto mišice. V primerjavi s sedemdnevno denervacijo je po štirinajstih dneh celokupna RNA v mišici statisticno znacilno (p<0.05) padla, zmanjšala pa se je tudi množina RNA na posamezno jedro.
ZAKLJUCKI: Ob predpostavki, da je sinteza dolocenih beljakovin ob posameznem jedru enaka, pomeni sedemnajstkrat vecja gostota jeder v plošcicnem, v primerjavi z zunajplošcicnim podrocjem, tudi sedemnajstkrat vecjo gostoto teh beljakovin v plošcicnem podrocju. To bi lahko bil pomemben dejavnik pri diferenciaciji plošcicnega dela mišicnega vlakna. Diferenciacija plošcicnega podrocja se odraža tudi v morfološki drugacnosti plošcicnih jeder v primerjavi z zunajplošcicnimi jedri. Nespremenjeni morfometricni parametri plošcicnih jeder po denervaciji kažejo bodisi na nepovratnost formiranja jedrnih skupkov, ali na prisotnost dejavnikov, ki vzdržujejo skupke jeder v nespremenjenem stanju tudi po dveh tednih denervacije. Povecano translacijsko kapaciteto na prostorninsko enoto denerviranega mišicnega vlakna lahko pripišemo zgošcevanju že obstojecih jeder in morda tudi povecanju celokupnega števila le-teh.
«»
[Abstract / English version]
Distribution of myonuclei and total extractable
RNA in normal and denervated fast muscle
Author: Tanja Milanic, Andrej Kunstelj
Mentor: Prof. dr. Zoran Grubic
INTRODUCTION: Apart from the majority of mammalian tissues, having mononuclear cell as a basic unit of the tissue composition, skeletal muscle fiber is a polynuclear syncytium. Polynuclear organization offers additional possibility for the regulation of protein expression: it can be organized at the level of myonuclear distribution and their specialization. Distribution of myonuclei can be considered as distribution of genes; their higher or lower concentration in the region may have important impact on their own expression, since higher or lower concentration of gene products could importantly control various stages of protein synthesis.
AIMS: The aim of our study was to analyse, whether morphomertical parameters of nuclear distribution in the normal and denervated muscle support the idea of regulation of gene expression at this level. The following hypothesies were tested:
1. Nuclear density in the motor endplate region exceeds extrajunctional nuclear concentration for at least one order of magnitude. At differences of this range one can expect important differences between both regions also at the level of gene product concentrations.
2. The sizes of nuclei in the endplate region are significantly different from the sizes of extrajunctional nuclei.
3. Nuclear clusters will disappear after denervation, because neural factors are essential for their organization.
4. Amount of total RNA belonging to individual nucleus will remain unchanged after denervation, because nuclear concentration and proliferation are the essential mechanisms responsible for increased total RNA in the muscle under such conditions.
METHODS: To test these hypothesies we developed a new technique based on the fluorescent staining of myonuclei and histochemical staining of motor endplates on the single isolated muscle fiber. Experiments were carried out on the rat sternomastoideus muscle. Image analyzer equipped with MCID (Micro Computer Imaging Device) was used in morphometric analyses of the fibers. Guanidinium-thiocyanate procedure was used for the isolation of total RNA. Unpaired t-test was applied at statistical analyses.
RESULTS: We found that synaptic concentration of myonuclei exceeds extrasynaptic concentration for the factor of 17. Longitudinal axis of the extrasynaptic nuclei was significantly (p<0.05) longer than the same axis of the synaptic nuclei. No significant changes in the number of nuclei accumulated at the endplates or in their morphometric criteria were observed after denervation, however, extrasynaptic nuclei became smaller (p<0.05) under such conditions. Seven days denervation resulted in insignificant (p>0.05) rise in total RNA per muscle and significantly increased total RNA per weight muscle, while total RNA per nucleus decreased. In comparison to seven days denervation, we observed significant (p<0.05) fall in total RNA per muscle and decreased total RNA per nucleus. We also found insignificant (p>0.05) fall in total RNA per weight muscle.
CONCLUSIONS: More than one order higher nuclear density in the endplate region means that at equal rate of protein synthesis at all myonuclei in the fiber, we can expect one order higher concentration of gene products in the synaptic region, which might be important for the differentiation of this region. Differences in gene expression between junctional and extrajunctional nuclei are also manifested at the level of their morphology. Unchanged morphometric parameters of the junctional nuclei after denervation are indicative either for ireversibility of the nuclear cluster formation or persistence of the factors responsible for their formation in the synaptic region after denervation. Concentration and proliferation of myonuclei was responsible for higher translational capacity in the muscle after denervation rather than increase in this capacity per nucleus.
|
|
 |
Išči po nalogah
Brskaj po nalogah
Prijava na obvestila
|
