Karakteristike poremećaja sinteze kolagena uzrokovane prenatalnom deprivacijom vitamina Ce kod zamorca

Abstract

LJudski organizam ima sposobnost sinteze kolagena, a jedan od ključnih momenata u njegovoj sintezi jeste proces hidroksilacije aminokiselina prolina i lizina za koju je neophodan vitamin Ce. U odrasloj dobi, deficit vitamina Ce usloviće poremećaj sinteze kolagena sa posledičnim razvojem skorbuta. Međutim, koje su to posledice poremećaja sinteze kolagena uzrokovane deficitom vitamina Ce tokom intrauterinog razvoja ploda još uvek su nedovoljno razjašnjene. Korišćenjem zamorca (Cavia porcelus) kao animalnog modela koji je genetski sličan čoveku u pogledu sinteze vitamina Ce, ispitan je uticaj prenatalne deprivacije ovog vitamina na sintezu kolagena i analizu promena nastalih u procesu organogenze. Eksperiment je dizajniran tako da su metodom slučajnog izbora ženke podeljene u tri grupe: kontrolnu (K) sa sedam skotnih ženki (n=7), i dve eksperimentalne E I (n=7) i E II (n=7). U kontrolnoj grupi sve vreme trajanja eksperimenta do 50-og dana gestacije ženke su dobijale vodu obogaćenu vitaminom Ce, dok u je E I grupi 10-og dana gestacije ženkama ukinut vitamin Ce, a u grupi E II 20-og gestacijskog dana. Ogled je koncipiran tako da je deprivacija vitamina Ce trajala 30 (E II), odnosno 40 dana (E I). Za histološku analizu plodovi su podvrgnuti transkardijalnoj perfuzionoj fiksaciji nakon koje su uzorkovana tkiva (tkivo bubrega, pluća, kosti, zuba, placente, velikog i malog mozga). Tkiva su procesuirana po standardnoj histološkoj proceduri i bojena imunohistohemijskim markerima. Za molekularnu i biohemijsku analizu nakon žrtvovanja uzorkovano je po 100 mg tkiva od interesa koja su uskladištena na -80ºC i čuvana do dalje analiza. Molekularna analiza obuhvatila je određivanje stepena ekspresije gena Coll1, Coll4a1, i Slc23a1, dok je biohemijska analiza obuhvatala određivanje koncentracije hidroksiprolina, kao kvantitativne mere količine kolagena. Na razvoj bubrega, prenatalni defiit vitamin Ce je imao veoma veliki uticaj. Eksperimentalni tretman je doveo do razvoja hipoplastičnog bubrega, koji je i po dimenzijama i po masi manji u odnosu na bubrege kontrolne grupe. Nefrogena zona je znatno šira u bubrezima iz grupe E I i E II, a analiza imunoeksprimiranosti ispitivanih marker govori u prilog usporenog sazrevanja. Eksperimentalni tretman je doveo do usporenog sazrevanja plućnog parenhima. Prema embriološkim fazama pluća su i do dve faze razvoja u zaostatku, što je samim tim rezultovalo i samanjenjem udela budućeg „vazdušnog“ prostora. Imunoekspresija ispitivanih markera potvrđuje navode vezane za zaostajanje u razvoju. Evidentan je uticaj nedostatka vitamina Ce tokom gestacije i na razvoj skeletne muskulature. Analizirajući histomorfološke karakteristike uočeno je da je veliki deo mišićnih vlakana u E I i E II grupi zadržao centralnu lokalizaciju jedara koje je ujedno većeg prečnika, što odgovara osobinama nezrelih miotuba. Imunohistohemijskim analizama nisu utvrđene bitnije razlike između analiziranih grupa. Na preparatima zuba plodova zamorca se kod vitamin Ce depriviranih jedinki registruje smanjena: gustina i dijametar kapilara zubne pulpe, debljina sloja predentina i dentina; prisutno je zaostajanja u procesu direktne osifikacije alveolarne kosti, kao i promene u imunoeskpresiji analiziranih markera. Prenatalni deficit vitamina Ce je na koštano-zglobnom sistemu plodova zamoraca doveo do izraženog smanjena ekstremiteta sa pristunim deformitetima; na histološkim preparatima je dokazano: drastično zaostajanje u enhondralnom okoštavanju, kod metakarpalne kosti dijafiza i metafiza od hrskavičavih ostrvaca bez znakova osifikacije, a međuprostor je ispunjen eritrocitima; epifiza je narušene kolumnarne organizacije sa dominacijom hipertrofičnih hondrocita, dok su proksimalne falange na nivou hrskavičavog modela sa početnim znacima stvaranja primarnog osifikacionog centra. Analizom količine hidroksiprolina u ispitivanim tkivima dokazano je u grupi E I povećanje količine hidroksiprolina u uzorcima tkiva bubrega, poprečno-prugastog mišića, velikog i malog mozga, placente i srca. Dok se u uzorcima pluća, kosti, zuba, hrskavice, plodovih ovojaka i tankog creva uočava smanjenje količine analiziranog parametra. U grupi E II veće količine hisroksiprolina su registrovane u uzorcima bubrega, poprečno-prugastog mišića, velikog i malog mozga, kosti, srca, placente i plodovih ovojaka. Sa druge strane smanjenje količine hisdroksiprolina je uočeno u uzorcima pluća, zuba, hrskavice, tankog creva i slezine. Analizom eksprimiranosti gena uočava se da je prenatalni deficit vitamina Ce doveo do povećanja eksprimiraosti Coll1 gena u uzorcima zuba, malog i velikog mozga. Eksperimentalni tretman je na uzorcima bubrega, pluća i poprečno-prugastog mišića doveo do smanjenja stepena eksprimiranosti analiziranog gena, dok u uzorcima koštanog tkiva nije utvrđena promena ekspresije. Ukidanje vitamina Ce je na uzorcima malog mozga i kosti dovelo do povećanja stepena eksprimiranosti Coll4a1 gena, dok je na plućima dobijeni rezultat smanjenjenje stepna eksprimiranosti. Deficit vitamina Ce nije doveo do promena eksprimiranostti Coll4a1 gena u uzorcima bubrega, poprečno-prugastog mišića, mozga i zuba. Prekid egzogenog unosa vitamina Ce tokom gestacije je na eksprimiranost Slc23a1 gena imalo uticaj u vidu povećanja stepena ekspresije na gotovo svim analiziranim organima (bubreg, pluća, mali i veliki mozak, zub i kost). Jedino u uzorcima poprečno-prugastog mišića nije utvrđena promena u stepenu ekspresije ispitivnog gena.

The human body can synthesize collagen, and one of the key moments in that process is the hydroxylation of the amino acids proline and lysine, for which vitamin C is necessary. In adults, vitamin C deficiency will disrupt collagen synthesis with the consequent development of scurvy. However, the consequences of disorders of collagen synthesis caused by vitamin C deficiency during intrauterine fetal development are still poorly understood. Using guinea pigs (Cavia porcelus) as an animal model that is genetically similar to humans in terms of vitamin C synthesis, the influence of prenatal deprivation of this vitamin on collagen synthesis as well as changes in organ development were investigated. During the experimentation, females were randomly divided into three groups: control (K) with seven pregnant females (n = 7), and two experimental E I (n = 7) and E II (n = 7). In the control group, females received water enriched with vitamin C throughout the experiment until the 50th day of gestation, while in the E I group on the 10th day of gestation, females received vitamin C, and in the E II group on the 20th day of gestation. The experiment was designed so that the deprivation of vitamin C lasted 30 (E II) and 40 days (E I). For histological analysis, fetuses were subjected to transcardiac perfusion fixation followed by tissue sampling (kidney, lung, bone, tooth, placental, cerebellar, and cerebellar tissues). Tissues were processed according to standard histological procedure and stained with immunohistochemical markers. For molecular and biochemical analysis after sacrifice, 100 mg of tissues of interest were sampled and stored at -80 °C and stored until further analysis. The molecular analysis included determining the degree of expression of the Coll1, Coll4a1, and Slc23a1 genes, while biochemical analysis included determining the concentration of hydroxyproline as a quantitative measure of collagen content. Prenatal vitamin C deficiency had a very large effect on kidney development. Experimental treatment led to the development of a hypoplastic kidney, which is smaller in size and weight compared to the kidneys of the control group. The nephrogenic zone is significantly wider in the kidneys from the E I and E II groups, and the analysis of the immunoexpression of the examined markers speaks in favour of delayed maturation. Experimental treatment led to delayed maturation of the lung parenchyma. According to the embryological phases of the lungs, up to two phases of development are lagging behind, which has resulted in a reduction in the share of future "air" space. Immunoexpression of the examined markers confirms the allegations related to developmental delay. The influence of vitamin C deficiency during gestation and on the development of skeletal muscles is evident. Analyzing the histomorphological characteristics, it was noticed that a large part of the muscle fibres in the E I and E II groups retained the central localization of the nucleus, which is also larger in diameter, which corresponds to the characteristics of immature myotubes. Immunohistochemical analyzes did not reveal significant differences between the analyzed groups. In guinea pig tooth preparations, the following is registered in vitamin C-deprived individuals: density and diameter of dental pulp capillaries, the thickness of predentine and dentin layers; there are delays in the process of direct ossification of the alveolar bone, as well as changes in the immunoexpression of the analyzed markers. Prenatal vitamin C deficiency on the skeletal system of guinea pig fruits led to a pronounced reduction of the extremities with present deformities; histological specimens have shown: drastic lag in enchondral ossification, in the metacarpal bone of the diaphysis and metaphysis of cartilaginous islets without signs of ossification, and the interstitial space is filled with erythrocytes; bone epiphysis is disturbed columnar organization with a predominance of hypertrophic chondrocytes, while the proximal phalanges are at the level of the cartilaginous model with the initial signs of the formation of the primary ossification centre. Analysis of the amount of hydroxyproline in the examined tissues showed an increase in the amount of hydroxyproline in samples of kidney, striated muscle, cerebellum, cerebellum, placenta and heart in group E I. While in the samples of lungs, bones, teeth, cartilage, fruit membranes and small intestine, a decrease in the amount of the analyzed parameter is observed. In the E II group, higher amounts of hydroxyproline were registered in samples of the kidney, striated muscle, cerebellum and cerebellum, bone, heart, placenta and amniotic sac. On the other hand, a decrease in the amount of hydroxyproline was observed in samples of the lungs, teeth, cartilage, small intestine and spleen. Analysis of gene expression shows that prenatal vitamin C deficiency has led to an increase in the expression of the Coll1 gene in the tooth, cerebellar, and cerebral samples. Experimental treatment on kidney, lung and striated muscle samples led to a decrease in the degree of expression of the analyzed gene, while no change in expression was found in bone tissue samples. Deprivation of vitamin C in cerebellar and bone samples led to an increase in the degree of expression of the Coll4a1 gene, while in the lungs the result was a decrease in the degree of expression. Vitamin C deficiency did not lead to changes in the expression of the Coll4a1 gene in samples from the kidneys, striated muscle, brain, and teeth. Discontinuation of exogenous vitamin C intake during gestation affected Slc23a1 gene expression in the form of increased expression on almost all analyzed organs (kidney, lung, cerebellum, cerebellum, tooth and bone). Only in the samples of the striated muscle, no change in the degree of expression of the analysed genes was found.