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DOI 10.26724/2079-8334-2018-4-66-198-202 UDC 611.018:616.311:57.017.642
PARTICIPATION OF INTESTINAL DIFFERENTIATION TRANSCRIPTION FACTOR CDX2 IN THE HISTOGENESIS OF THE GASTROINTESTINAL TRACT
E-mail: tatuana.rekun@gmail.com
The purpose of the present study was to explore expression and role of CDX2 in the differentiation of anterior and posterior parts of the gastrointestinal tract in the human embryos and fetuses in gestation age 4-38 weeks. The current study was carried out on 169 autopsied formalin-fixed human embryos and fetuses of gestational age 4-38 weeks of Caucasian ethnic origin. It was found that expression of CDX2 was negative in all cases as in esophageal so in gastric epithelium, despite its moderate expression in small and large intestine. This indicates that CDX2 is important transcription factor of development and differentiation of exactly intestinal epithelium. According to this view, immunohistochemical and functional experimental investigations point to appropriate CDX2 expression during the Barrett's esophagus formation and on the contrary conditional removal of CDX2 in the intestine causes squamous metaplasia. Expression of intestinal differentiation transcription factor CDX2 was typical for epithelial differon of lower parts of the gastrointestinal tract and wasn't noticed by us in the enterocytes nuclei of the esophagus and stomach.
Key words: embryogenesis, transcription factor CDX2, gastroesophageal and intestinal epithelium, gastrointestinal tract.
The present study is a fragment of the research project "Early diagnostics of dysplastic, metastatic and neoplastic changes in pathology of the gastrointestinal tract, respiratory, genitourinary and neuroendocrine systems" (state registration No. 0117U000001).
It is known that during early embryogenesis the gastrointestinal tract (GIT) develops from the two endodermal invaginations, namely from anterior part of the primary gut and posterior caudal part of the primary gut. Certain transcription factors express in the endoderm of anterior part of the primary gut and some in the caudal part [1, 7]. Different molecular ways and transcription factors were described in the process of esophagus, stomach, small and large intestine development. However many questions regarding specification (specialization and determination) of the gut endoderm remain to be addressed.
It is thought that gut endoderm differentiate locally at early stages and specification determines by the interaction with surrounding mesenchyme [4, 12]. There are next key mechanisms which influence differentiation of the GIT: BMP (bone morphogenetic protein), proteins of signal ways Sonic Hedgehog (Shh) and Notch, transcription protein P63, CDX1 and CDX2 (intestinal differentiation factors). Transcription factors HDX, SOX, Foxa2 that always express and are typical for endoderm, signal systems Wnt and TCP signal ways [2, 8, 14]. Aberrant expression of CDX2 in the upper parts of the GIT can be important pathogenic element of the intestinal metaplasia of the stomach mucosa and Barrett's esophagus.
Despite a great number of studies, participation of transcription factors in the epithelium differentiation, its renewal and homeostasis is complicated and insufficiently researched. Molecular mechanisms of the stratified squamous epithelium transformation into the columnar one are still unclear.
The purpose of research was to investigate expression and role of CDX2 in the differentiation of anterior and posterior parts of the GIT in the human embryos and fetuses in gestation age 4-38 weeks.
Materials and methods. The current study was carried out on 169 autopsied formalin-fixed human embryos and fetuses of gestational age 4-38 weeks of Caucasian ethnic origin. Miscarriage tissue was collected through dilatation and curettage procedure. No suction was performed to collect this tissue. The fresh specimens were evaluated by pathologist in order to isolate the embryo or fetal component in the miscarriage tissue. The criteria for a macroscopic identification of the embryo or fetal component were next: the embryo or fetal shape, denser pattern and a color that tends toward a translucent grey. All autopsies were performed between 4 and 24 h after death under standard protocols on the base of Vinnytsia regional pathoanatomical bureau. All mothers provided a written consent to the anonymous use of their data for research purposes. This consent includes last name and first name of the mother, age of the mother, diagnosis of the mother, sex of the fetus, gestation age and diagnosis of the fetus. Condition of the fetus was evaluated by anthropometric indices, data of external and internal investigation. The presence of congenital malformations involving the GIT was considered as an exclusion criterion. Crown-rump length (CRL) was the main criterion for estimating fetal age. The total of embryos and fetuses were 169. They were divided by age and CRL into next groups (table 1).
Histological methods of investigation include: hematoxylin and eosin staining, alcian blue staining and periodic acid-Schiff reaction (PAS). Immunohistochemical studies were performed on paraffin
© T.O. Rekun, Yu.Yu. Trofimenko, 2018 198
sections using streptavidin-biotin method ("DAKO", Denmark, LSAB2 Systems, HRP). Antigen unmasking was carried out in citrate buffer with pH 6,0. Mice and rabbits monoclonal antibodies were used as primary antibodies. Cell nuclei were stained with Mayer's hematoxylin for 15-60 seconds. The index of the intestinal differentiation and nuclear markers CDX2 were determined in five randomly selected fields of vision ( > 500 cells) as a percentage of positively stained nuclei of the epithelial cells of the esophagus, stomach and intestine in the preparations under 400x magnification of the microscope. All patients were thoroughly informed about research, which was approved by the bioethics committee.
Table 1
Distribution of human embryos and fetuses into age groups
№ Age (weeks) Number CRL, mm (M±a)
1 4-5 6 6.065±0.030
2 6-7 6 15.12±0.63
3 8-9 7 19.43±0.52
4 10-11 10 39.02±0.51
5 12-13 11 58.72±2.27
6 14-15 12 93.11±5.11
7 16-17 15 122.2±2.7
8 18-20 19 152.9±3.5
9 21-24 20 192.1±1.8
10 25-28 13 231.0±3.1
11 29-32 14 264.8±1.7
12 33-36 20 302.9±1.4
13 37-38 16 341.5±5.7
Total 169
Statistical analysis was performed using software Microsoft Office Excel 2003 and "Statistica 5.0" using nonparametric methods.
Results of the study and their discussion. In embryos with CRL 6.065 ± 0.030 mm (with gestational age 4-5 weeks) and 15,12 ± 0,63 mm (gestational age 6-7 weeks) the esophageal epithelium consisted of two or three cells thick layer, herewith the deepest layer was formed by columnar cells (stratum basale). Until eight weeks with CRL 19,43 ± 0.52 mm the epithelium remained pseudostratified with appearance of single vacuoles in the cell cytoplasm. After 10 weeks (CRL 39,02 ± 0,51 mm) the epithelium became stratified columnar. The gastric epithelium of the gastroesophageal junction resembles esophageal one according histochemical characteristics at the given period of observation, but this epithelium had more compact nuclei arrangement with their hyperchromasia. There was three-layered or four-layered epithelium arrangement, sometimes chaotic, with majority of elongated nuclei. Already in this period, we observed the appearance of single vacuoles as in subnuclear so in supra nuclear compartments, appearance of PAS positive border was typical in some places, which also was noticed under hematoxylin and eosin specimen staining. Near esophageal and gastric epithelium of the gastroesophageal junction, crimson stained granules in the cytoplasm around nuclei of small intestine goblet cells were noted in the fetuses with gestational age 11-12 weeks. This staining was well noticeable while using the PAS (figure 1), which was the evidence of the presence of neutral mucins in the intestinal epithelium cytoplasm and unite epithelial differons of upper and lower parts of the GIT by histochemical properties. Expression of transcription factor CDX2 wasn't noticeable at the given period of observation in the epithelial differon of the esophagus, stomach and intestine (figure 2).
Fig. 1. Neutral mucins (1) in the cytoplasm of the small Fig. 2. Absence of the intestinal differentiation transcription
intestine goblet cells. Small intestine mucosa of the fetus, gestational factor expression in nuclei of the gastroesophageal junction age - 12 weeks. PAS, x 200. epithelium in the fetus with gestational age - 11 weeks.
Immunohistochemical reaction with CDX2 antibody, x 100.
It should be noted that CDX2 expression in nuclei of the columnar epithelium of different parts of the small and large intestine started from 16 weeks CRL 122.2 ± 2.7 mm and appeared until 38 weeks CRL 341.5 ± 5.7 mm and wasn't revealed in early embryonal and fetal periods of prenatal ontogenesis. After 16 weeks, enterocytes of the small intestine were positive to CDX2 and in the majority of cases were shown homogenous staining (figure 3). Moderate nuclear staining was observed in the enterocytes of the large intestine (figure 4).
Fig. 3. A strong expression of transcription factor CDX2 in Fig. 4. A moderate expression of transcription factor
nuclei of the small intestine enterocytes in the fetus with gestational age CDX2 in the nuclei of the large intestine enterocytes in the fetus
- 11 weeks. Immunohistochemical reaction with CDX2 antibody, with gestational age - 11 weeks. Immunohistochemical reaction
x 200. with CDX2 antibody, x 200.
It is important to notice that after 16 weeks of gestation enterocytes nuclei of the small and large intestine were positive to CDX2 with focal staining in 95% of cases. CDX2 expression didn't express in the esophagus and stomach mucosa in comparison with small and large intestine at the given period of observation.
Nowadays special attention deserves investigations which connect participation of transcription factors during appearance of Barrett's esophagus and neoplastic changes of the GIT mucosa. Some theories were brought up including transdifferentiation of one committed cell into another or transformation of population of progenitor cells or stem cells into simple epithelium instead of stratified squamous non keratinized which physiologically lines esophagus [9, 14]. From molecular point of view transcription factor CDX2 is one of the key links. It is known that esophagus develops from anterior gut endoderm. During embryonal development esophageal epithelium subjects to a number of changes which include differentiation of stem cells into different epithelial types. The GIT development can be divided into periods where epithelium is changing constantly. This connects with different molecular ways that regulate development of the GIT. Important to notice that certain transcription factors express during different periods of embryogenesis. Starting from eighth week of gestational age esophageal epithelium is changing into ciliated (resemble respiratory epithelium) than squamous (resemble epithelium of the lower parts of the GIT) and finally into stratified squamous non keratinized epithelium.
According to our investigation CDX2 expression was negative in all cases as in esophageal so in gastric epithelium, despite its moderate expression in small and large intestine. This indicates that CDX2 is important transcription factor of development and differentiation of exactly intestinal epithelium. According to this view, immunohistochemical and functional experimental investigations point to appropriate CDX2 expression during the Barrett's esophagus formation and on the contrary conditional removal of CDX2 in the intestine causes squamous metaplasia.
Completely differentiated squamous epithelium could undergo phenotypical changes through molecular programming into intestinal columnar cells without mitosis. This process is called transdifferentiation. As known, this process underlies a lot of pathological processes, for example the Barrett's esophagus. Special attention deserves source of origin for cells ant tissues from which metaplastic epithelium forms. Sources of origin include: proper differentiated cells of the stratified squamous non keratinized epithelium; local undifferentiated cells that locate in the epithelium of the esophagus or in the ducts of the glands; migrating stem cells from red bone marrow or columnar cells from the gastroesophageal junction or cardiac region of the stomach, that proximally transfer into esophagus replacing squamous cells [3, 10, 11]. Transformation of stratified squamous epithelium of the esophagus can include transitional period of columnar epithelium. This is probably provided through reprogramming of basal cells from glands duct or cells from gastroesophageal junction, as a result of which CDX2 expression occurs that causes intestinal metaplasia. Phenotypical replacement of squamous epithelium into columnar one includes a number of genetic alterations. Numerous researches indicate that different
transcription factors express in different periods of the GIT development [5, 7, 15]. Completely differentiated cells of the gastrointestinal junction may be subject to transdifferentiation namely transformation of one type of epithelium into another without mitotic division. Still it remains unclear whether this differon (histogenetic cell lineage) includes necessary precursor cell of stratified squamous epithelium or stem cell with ability to become columnar cell of the intestine.
Thereby data received in our research coincide with investigations of Tsutomu Mizoshita et al. (2001) and Coskun M. et al. (2011) [2, 6, 7, 13], that CDX2 expression in human limit oneself solely with small and large intestine, within the boundaries from duodenum to rectum.
Expression of intestinal differentiation transcription factor CDX2 was typical for epithelial differon of lower parts of the GIT and wasn't noticed by us in the enterocytes nuclei of the esophagus and stomach.
Further study of the newest transcription factors that participate in formation and differentiation of the GIT cell differons opens perspectives for deeper understanding of pathogenic chains transformation of different epithelial types of the gastroesophageal junction as at different stages of the embryogenesis so during appearance of precancerous changes in the pathology.
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УЧАСТЬ ТРАНСКРИПЦ1ИНОГО ФАКТОРУ КИШКОВО1 ДИФЕРЕНЩАЦП CDX2 У Г1СТОГЕНЕЗ1 ШЛУНКОВО-КИШКОВОГО ТРАКТУ Рекун Т.О., Трофiменко Ю.Ю., Балинська М.В., Лобода 1.В. Метою роботи було дослщити експреаю та роль CDX2 у диференщюванш верхньо! та нижньо! частини шлунково-кишкового тракту у людських ембрюшв та плодiв у термш гестацп 4-38 тижшв. Дослщження включало 169 аутопсшних зафксованих у формалш людських ембрюшв та плодiв у термш гестацп 4-38 тижшв европейського етшчного походження. Встановлено, що експреая CDX2 була негативною у вЫх випадках як в стравохщному, так i в шлунковому ештелто, попре помiрну експреаю його в тонкш та товстш кишщ, що свщчить про те, що CDX2 являеться важливим транскрипцшним фактором розвитку та
УЧАСТИЕ ТРАНСКРИПЦИОННЫХ ФАКТОРОВ КИШЕЧНОИ ДИФФЕРЕНЦИАЦИИ CDX2
В ГИСТОГЕНЕЗЕ ЖКТ Рекун Т.А., Трофименко Ю.Ю., Балинская М.В., Лобода И.В.
Целью работы было исследовать экспрессию и роль СЭХ2 в дифференцировании верхней и нижней части желудочно-кишечного тракта человеческих эмбрионов и плодов в сроке гестации 4-38 недель. Исследование включало 169 аутопсий, зафиксированных в формалине человеческих эмбрионов и плодов в сроке гестации 4-38 недель европейского этнического происхождения. Установлено, что экспрессия CDX2 была отрицательной во всех случаях как в пищеводном, так и в желудочном эпителие, показало умеренную экспрессию его в тонкой и толстой кишке, что свидетельствует о том, что CDX2 является важным транскрипционных факторов развития и дифференциации именно кишечного эпителия. Согласно
диференщацп саме кишкового еттелш. Вщповщно до ще! точки зору iмуногiстохiмiчнi та функщональш експериментальнi дослiдження вказують на вщповщну експресiю СБХ2 при утворенш стравоходу Барретта, i навпаки умовне видалення СБХ2 в кишцi призводить до сквамозно! метаплазп. Експресiя транскрипцшного фактору кишково! диференщацп СБХ2 була характерна для еmтелiального диферону нижшх вiддiлiв шлунково-кишкового тракту та не спостерталася нами в ядрах еттелюциив стравоходу та шлунка.
Ключовi слова: ембрюгенез, транскрипцiйний фактор СБХ2, гастроезофагеальний та кишковий еттелш, шлунково-кишковий тракт.
Стаття надiйшла: 5.06.18 р.
этой точке зрения иммуногистохимические и функциональные экспериментальные исследования указывают на соответствующую экспрессию СБХ2 при образовании пищевода Барретта, и наоборот условное удаления СБХ2 в кишке приводит к сквамозной метаплазии. Экспрессия транскрипционного фактора кишечной дифференциации СБХ2 была характерна для эпителиального дифферона нижнего отдела желудочно-кишечного тракта, но не наблюдалась нами в ядрах эпителиоцитов пищевода и желудка.
Ключевые слова: эмбриогенез, транскрипционный фактор СБХ2, гастроэзофагеальный и кишечный эпителий, желудочно-кишечный тракт.
Рецензент Шеттько В.1.
DOI 10.26724/2079-8334-2018-4-66-202-207 UDC 611.835.8-031:615.368]-07-092.9
ХАРАКТЕРИСТИКА СТРУКТУРНИХ КОМПОНЕНТ1В СТОВБУРА С1ДНИЧНОГО НЕРВА ПРИ ВВЕДЕНН1 КРЮКОНСЕРВОВАНО! ПЛАЦЕНТИ У ЩУР1В
E-mail: Shepitko1973@ukr.net
Ушкодження переферичних нерв!в рiзними чинниками е актуальною проблемою сучасно! практично! та експериментально! медицини. При вpажeннi запальним процесом перефершних нepвiв, лiкування, та реабштащя таких хворих займае тривалий час. Метою дослщження було встановлення змш морфометричних паpамeтpiв структурних елеменив стовбура сiдничного нерва у щуpiв при одноразовому пiдшкipному ввeдeннi крюконсервовано! плаценти. Стовбур сiдничного нерва був вилучений вщ 50 статeвозpiлих щуpiв-самцiв лiнii' "Вiстаp". За допомогою мкроскопу з цифровою мiкpофотонасадкою проводили морфометрш наступних показникiв: загальна товщина Ыдничного нерва, товщина eпiнeвpiю, товщина периневрш, товщина eндонeвpiю. Виявлено, що показники загально! товщини, eпiнeвpiю та ендоневрто стовбура сiдничного нерва щуpiв реагували збшьшенням показника з максимальним значенням на 3-у добу експерименту та повним вщновленням !х паpамeтpiв на 7-му добу. Встановлено, що вщновлення вах показникiв до значень штактно! групи було виявлено на 7-у добу експерименту. Вищевикладене припускае, що ноpмалiзацiя всiх морфометричних показникiв Ыдничного нерва на 7 добу може бути тшьки пiсля одноразового введення крюконсервовано! плаценти.
IGii040Bi слова: стовбур, сщничний нерв, крюконсервована плацента, експеримент.
Робота е фрагментом НДР «Експериментально-морфологiчне вивчення ди mpaHcmaHmamie ^оконсервовано'1 плаценти на морфофункщональний стан ряду внутрштх оргатв» № державно'1 реестрацн 0113U006185.
Проблема д1агностики та лшування захворювань периферично! нервово! системи залишаеться актуальною темою ХХ1 стол1ття. Враховуючи, що захворювання периферично! нервово! системи нерщко виникають у ос1б р1зного вшу, а переважно у ос1б працездатного вшу, лшування !х потребуе значних матер1альних витрат, як з боку хворих так i з державних джерел, що вказуе на соцiально-eкономiчну важливiсть дано! проблеми [1]. При цьому необхщно вiдмiтити, що загальна нейротравматизащя в середньому зростае на 2% [2, 13]. Щц час вшськових дiй цей показник становить 12% i спостepiгаеться у 2,8-5% пащенпв з полiтpавмою, переважна частина яких е вшськовослужбовщ [2].
Кшець ХХ столiття вiдзначився новими методами лшування захворювань пов'язаних з порушенням iмунно-обмiнних пpоцeсiв, регенераторно! функци [8, 12]. Одним з таких був визнаний метод корекци запальних пpоцeсiв за допомогою тканинно^нженерного пiдходу, введення в оpганiзм препарапв бiологiчного походження [5], а саме - введення крюконсервовано! плаценти, тканини та кттини яко! мiстять бюлопчно активнi речовини i впливають на iмунну систему як модулятор [9].
Низкою автоpiв були здiйснeнi фундамeнтальнi дослiджeння в цш галузi. Було сформовано цше направлення в вивчeннi взаемовiдносин мiж живим оpганiзмом i бiологiчно активними живими клггинами [4, 6, 7, 9, 10, 11].
У зв'язку з цим, без сумшву, защкавлешсть викликае використання методу введення фрагменту крюконсервовано! плаценти для корекцй ушкоджено! периферично! нервово! системи.
© Р.В. Свиридюк, В.1. Шеттько, 2018
