REUTILIZATION OF LYMPHOCYTE DNA BY CELLS OF INTESTINAL CRYPTS AND REGENERATING LIVER

Lymphoid cells from mice injected 54 hours and 30 hours earlier with ³H-thymidine were washed and transfused into isogenic recipients at 29 to 30 hours after partial hepatectomy. The recipients were killed 28 to 30 hours later, and liver, intestine, Peyer''s patch, spleen, and the transfused cells were examined in autoradiographs exposed 6 months. Approximately 80 per cent of the labeled transfused cells were classed as lymphocytes. The labeled DNA contained in the transfused cells was partitioned to about 14 times as many recipient liver and intestinal cells, appearing in 72 to 78 per cent of hepatocyte nuclei, in 30 to 35 per cent of liver reticuloendothelial nuclei, and in 90 to 95 per cent of intestinal crypt nuclei. The label was not comparably widespread in the lymphoid organs, but was limited to a few intensely labeled lymphocytes and a somewhat larger number of very weakly labeled cells. When heat-killed cells rather than living cells were transfused, intensely labeled lymphocytes were absent from the lymphoid organs, but the labeling of cells in the recipients was otherwise identical. The results suggest that (a) reutilized DNA is derived from dead cells, (b) reutilized DNA is mainly degraded to nucleosides and nucleotides, the usual immediate de novo DNA precursors, before reincorporation into DNA, and (c) DNA reutilization may occur in the lymphoid organs, but on a less active scale than in intestine or regenerating liver.     

Existence of serotonin and neuropeptides-immunoreactive endocrine cells in the small and large intestines of the mole-rats (Spalax leucodon)

The present study was conducted to clarify the regional distribution and relative frequency of endocrine cells secreting serotonin, substance P (SP), cholecystokinin-8 (CCK-8), vasoactive intestinal polypeptide (VIP) and neurotensin in the small and large intestine of the mole-rats (Spalax leucodon), by specific immunohistochemical methods. In the small and large intestine of mole-rats (Spalax leucodon), serotonin, SP and VIP were identified with various frequencies, but CCK-8 and neurotensin were not observed. Most of the IR cells in the small and large intestine were located in the intestinal crypt and epithelium however, they were more frequency in the intestinal crypt. Serotonin-IR cells were detected throughout the whole intestinal tract, predominantly in the duodenum and colon. SP-IR cells were demonstrated throughout the whole intestinal tract except for the ileum and rectum with highest frequencies in the cecum. VIP-IR cells were found in all parts of the small intestine except for the large intestine.In conclusion, the general distribution patterns and relative frequency of intestinal endocrine cells of the mole-rats (Spalax leucodon) was similar to those of some rodent species. However, some species-dependent unique distributions and frequencies characteristics of endocrine cells were also observed in the present study.     

RNA synthesis by villus and crypt cell nuclei of rat intestinal mucosa

Rat intestinal mucosa was separated by eversion and vibration to provide a sequence of fractions from predominantly villus cells to predominantly crypt cells. The proportions of these cell types in each fraction were computed from the concentrations of alkaline phosphatase (villus cells) and thymidine kinase (crypt cells) in each population. The isolated mucosal fractions varied from about 90% villus cells to 90% crypt cells. Following injection of the rats with [³H]thymidine, the nuclei were isolated from each mucosal cell fraction and the amount of radioactivity incorporated into DNA was measured as an index of crypt cell abundance. The isolated nuclei were also incubated with ribonucleoside triphosphates and the amount of RNA synthesized was measured. Nuclei labeled with [³H]thymidine were found only in fractions rich in crypt cells, whereas capacity for RNA synthesis remained very active in mucosal fractions consisting predominantly of villus cells. It is concluded that non-dividing villus cells continue to make RNA.     

Thyroid Hormone Regulation of Adult Intestinal Stem Cell Development: Mechanisms and Evolutionary Conservations

The adult mammalian intestine has long been used as a model to study adult stem cell function and tissue renewal as the intestinal epithelium is constantly undergoing self-renewal throughout adult life. This is accomplished through the proliferation and subsequent differentiation of the adult stem cells located in the crypt. The development of this self-renewal system is, however, poorly understood. A number of studies suggest that the formation/maturation of the adult intestine is conserved in vertebrates and depends on endogenous thyroid hormone (T3). In amphibians such as Xenopus laevis, the process takes place during metamorphosis, which is totally dependent upon T3 and resembles postembryonic development in mammals when T3 levels are also high. During metamorphosis, the larval epithelial cells in the tadpole intestine undergo apoptosis and concurrently, adult epithelial stem/progenitor cells are formed de novo, which subsequently lead to the formation of a trough-crest axis of the epithelial fold in the frog, resembling the crypt-villus axis in the adult mammalian intestine. Here we will review some recent molecular and genetic studies that support the conservation of the development of the adult intestinal stem cells in vertebrates. We will discuss the mechanisms by which T3 regulates this process via its nuclear receptors.     

THE EFFECT OF INTESTINAL RESECTION ON THIRY-VELLA FISTULAE OF JEJUNAL AND ILEAL ORIGIN IN THE RAT: EVIDENCE FOR A SYSTEMIC CONTROL MECHANISM OF CELL RENEWAL

Local and systemic control mechanisms have been postulated to explain the maintenance of steady state cell renewal in intestinal epithelium. Permanent alterations of cell renewal resulting in a new steady state imply alterations in control. Intestinal resection appears to cause such alterations resulting in hyper-plasia of the residual intestine. To test the hypothesis of a systemic control, the effect of 60% mid-intestinal resection on Thiry-Vella fistulae of both jejunal and ileal origin was observed in rats. Results showed that hypoplasia occurred in fistulae without resection of the remaining intestine in continuity. Cell counts of crypt and villus columns and tritiated thymidine uptake in isolated whole crypts were reduced. Scanning electron microscopy showed marked hypoplastic alterations in villi. However, when 60% of the intestine in continuity was resected, hyperplasia occurred not only in the residual intestine but in the fistulae of both jejunal and ileal origin. Cell counts of villus and crypt columns were increased along with increased tritiated thymidine uptake per crypt. Neutral cc-glucosidase and non-specific esterase activities did not change as a result of resection but the activities of both enzymes were greater in ileal fistulae than in ileum in situ. Observations on the different resection response of the jejunal versus ileal fistulae lead to a distinction between inherent and induced differences within the small intestine. This study suggests a systemic control of cell renewal. A possible mechanism involving intestinal vascular physiology is discussed.     

Spatio-temporal expression profile of stem cell-associated gene LGR5 in the intestine during thyroid hormone-dependent metamorphosis in Xenopus laevis

Background

The intestinal epithelium undergoes constant self-renewal throughout adult life across vertebrates. This is accomplished through the proliferation and subsequent differentiation of the adult stem cells. This self-renewal system is established in the so-called postembryonic developmental period in mammals when endogenous thyroid hormone (T3) levels are high.

Methodology/Principal Findings

The T3-dependent metamorphosis in anurans like Xenopus laevis resembles the mammalian postembryonic development and offers a unique opportunity to study how the adult stem cells are developed. The tadpole intestine is predominantly a monolayer of larval epithelial cells. During metamorphosis, the larval epithelial cells undergo apoptosis and, concurrently, adult epithelial stem/progenitor cells develop de novo, rapidly proliferate, and then differentiate to establish a trough-crest axis of the epithelial fold, resembling the crypt-villus axis in the adult mammalian intestine. The leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a well-established stem cell marker in the adult mouse intestinal crypt. Here we have cloned and analyzed the spatiotemporal expression profile of LGR5 gene during frog metamorphosis. We show that the two duplicated LGR5 genes in Xenopus laevis and the LGR5 gene in Xenopus tropicalis are highly homologous to the LGR5 in other vertebrates. The expression of LGR5 is induced in the limb, tail, and intestine by T3 during metamorphosis. More importantly, LGR5 mRNA is localized to the developing adult epithelial stem cells of the intestine.

Conclusions/Significance

These results suggest that LGR5-expressing cells are the stem/progenitor cells of the adult intestine and that LGR5 plays a role in the development and/or maintenance of the adult intestinal stem cells during postembryonic development in vertebrates.     

CONTROL OF INTESTINAL EPITHELIAL REPLACEMENT: LACK OF EVIDENCE FOR A TISSUE-SPECIFIC BLOOD-BORNE FACTOR

The small intestine of rats was cut across in two places, about 14 and 50% of the length of the small intestine from the pylorus, and continuity was re-established by suturing the proximal and distal ends. The resulting sac of small intestine, averaging 36% of the total length of the small intestine, had its upper end closed off, and its lower end anastomosed, either to the intestine-in-continuity (an ‘intestine-sac’), or to the skin of the abdominal wall (a ‘skin-sac’). On the ninth post-operative day, the cell production rate in squashes of micro-dissected whole crypts of Lieberkühn was measured by mitotic blockade with Colcemid. The rate of cell production in unoperated and sham-operated rats was 30 cells/crypt/hr, throughout the length of the small intestine. In the intestine in continuity, the rate increased to an average of 46 cells/crypt/hr above the anastomosis, and to 54 cells/crypt/hr below it. At the lower end of the ‘intestine-sac’, which drained into the intestine-in-continuity, the rate was 39 cells/crypt/hr, while in the lower end of the sac which drained to skin the rate of cell production was only 16 cells/crypt/hr. This significantly lower cell production rate in intestine which was not in contact with ingesta is taken to be evidence of the importance of local, rather than blood-borne factors in the control of epithelial replacement.     

Intestinal epithelial stem/progenitor cells are controlled by mucosal afferent nerves

Background

The maintenance of the intestinal epithelium is of great importance for the survival of the organism. A possible nervous control of epithelial cell renewal was studied in rats and mice.

Methods

Mucosal afferent nerves were stimulated by exposing the intestinal mucosa to capsaicin (1.6 mM), which stimulates intestinal external axons. Epithelial cell renewal was investigated in the jejunum by measuring intestinal thymidine kinase (TK) activity, intestinal ³H-thymidine incorporation into DNA, and the number of crypt cells labeled with BrdU. The influence of the external gut innervation was minimized by severing the periarterial nerves.

Principal Findings

Luminal capsaicin increased all the studied variables, an effect nervously mediated to judge from inhibitory effects on TK activity or ³H-thymidine incorporation into DNA by exposing the mucosa to lidocaine (a local anesthetic) or by giving four different neurotransmitter receptor antagonists i.v. (muscarinic, nicotinic, neurokinin1 (NK1) or calcitonin gene related peptide (CGRP) receptors). After degeneration of the intestinal external nerves capsaicin did not increase TK activity, suggesting the involvement of an axon reflex. Intra-arterial infusion of Substance P (SP) or CGRP increased intestinal TK activity, a response abolished by muscarinic receptor blockade. Immunohistochemistry suggested presence of M3 and M5 muscarinic receptors on the intestinal stem/progenitor cells. We propose that the stem/progenitor cells are controlled by cholinergic nerves, which, in turn, are influenced by mucosal afferent neuron(s) releasing acetylcholine and/or SP and/or CGRP. In mice lacking the capsaicin receptor, thymidine incorporation into DNA and number of crypt cells labeled with BrdU was lower than in wild type animals suggesting that nerves are important also in the absence of luminal capsaicin, a conclusion also supported by the observation that atropine lowered thymidine incorporation into DNA by 60% in control rat segments.

Conclusion

Enteric nerves are of importance in maintaining the intestinal epithelial barrier.     

A METHOD FOR QUANTITATION OF PROLIFERATIVE INTESTINAL MUCOSAL CELLS ON A WEIGHT BASIS: SOME VALUES FOR C57BL/6

A method for determining the number of intestinal mucosal crypts, S cells, and total proliferative cells, on a weight basis has been presented. The number of crypts was obtained (following injection of tritiated thymidine) by dividing the disintegrations per minute (dpm) per mg intestine by the dpm per crypt. Multiplication of the number of crypts per mg by the number of labeled cells per crypt (determined radioautographically) resulted in the number of S cells per mg intestine. Division of the number of S cells per mg by the fraction of proliferative cells in S (obtained by cell cycle analysis) resulted in the number of proliferative cells per mg intestine. Values for duodenum, jejunum, and ileum of male C57BL/6 mice are given.     

EFFECT OF VILLUS LENGTH ON CELL PROLIFERATION AND MIGRATION IN SMALL INTESTINAL EPITHELIUM

For the interpretation of data supporting the hypothesis of a feedback regulation of proliferative activity in intestinal crypts by the functional villus cell compartment the life span and migration rate of epithelial cells on villi of experimentally reduced length should be known. Autoradiographic studies and scintillation counting of isolated villi at different time intervals after ³H-thymidine labelling were carried out 36, 48 and 60 hr intervals after X-irradiation. The results showed that the life span of epithelial cells in rat small intestine (36–48 hr) is independent of the villus length. In villi of reduced length the migration rate of the epithelial cells was found to be decreased compared with controls. Changes in the migration rate in turn seem to be dependent on the production of epithelial cells in the crypt. Comparative studies on the recovery of crypt and villus epithelium after various doses (300 and 700 R) of X-radiation support the hypothesis that increased proliferative activity in the crypt cell compartment is related to a reduction of the number of functional villus cells below a critical villus length. The importance of these findings in the interpretation of data on (micro) biochemical analyses of certain cell differentiation characteristics during increased proliferative activity is discussed.     

Recent progress in the study of brown adipose tissue

Adult organ-specific stem cells are essential for organ homeostasis and repair in adult vertebrates. The intestine is one of the best-studied organs in this regard. The intestinal epithelium undergoes constant self-renewal throughout adult life across vertebrates through the proliferation and subsequent differentiation of the adult stem cells. This self-renewal system is established late during development, around birth, in mammals when endogenous thyroid hormone (T3) levels are high. Amphibian metamorphosis resembles mammalian postembryonic development around birth and is totally dependent upon the presence of high levels of T3. During this process, the tadpole intestine, predominantly a monolayer of larval epithelial cells, undergoes drastic transformation. The larval epithelial cells undergo apoptosis and concurrently, adult epithelial stem/progenitor cells develop de novo, rapidly proliferate, and then differentiate to establish a trough-crest axis of the epithelial fold, resembling the crypt-villus axis in the adult mammalian intestine. We and others have studied the T3-dependent remodeling of the intestine in Xenopus laevis. Here we will highlight some of the recent findings on the origin of the adult intestinal stem cells. We will discuss observations suggesting that liganded T3 receptor (TR) regulates cell autonomous formation of adult intestinal progenitor cells and that T3 action in the connective tissue is important for the establishment of the stem cell niche. We will further review evidence suggesting similar T3-dependent formation of adult intestinal stem cells in other vertebrates.     

Calpain from rat intestinal epithelial cells: Age-dependent dynamics during cell differentiation

Micromolar and millimolar Ca²⁺-requiring neutral protease (calpain I and calpain II) along with their endogenous inhibitor calpastatin were isolated and partially purified from the same preparation of rat intestinal epithelial cells. Calpain I and II were partially purified by 1300 and 900-fold with 57 and 53 per cent yield, respectively. The optimum assay conditions revealed pH 7.5, 20 min incubation at 25° C and 0.24% casein substrate for both calpains. The optimum calcium concentration obtained for calpain I and II were 25 M and 4 mM, respectively. Distribution of rat intestinal epithelial cells calpain I and II along with calpastatin during cell differentiation stages in weanling to senescence age were studied. Calpain I in weanling rats was in an increasing order from villus to crypt regions. Adult rats indicated well expressed consistent calpain I throughout the differentiation stages. Whereas, significant lowering towards crypt region cells were evident in old rats. Calpain II in weanling and adult rats was found to be consistent throughout the differentiation stages. Old animals revealed an increasing trend from villus to crypt region with insignificant activity present in upper villus cells. Concomitantly, different concentrations of calpastatin were observed throughout the differentiation stages in all the age groups. Moreover, the levels of calpains exceeded that of calpastatin in most of the epithelial cell populations during developmental stages. In addition to casein, intestinal epithelial cell membranes were found to be equally good substrates for calpains. Proteolytic susceptibility of weanling, adult and old rat membrane proteins varied significantly all along the ageing process in rats. Simultaneous age-dependent calpastatin response were also evident. Taken together the results obtained provided strong evidence that calpain plays significant role in rat intestinal cell differentiation and ageing process with calpastatin as its specific regulatory protein.Abbreviations DEAE-cellulose O-(Diethylaminoethyl)-cellulose - EDTA Ethylene Diamine Tetra Acetic Acid - Tris Tris (hydroxymethyl) amino methane - KH₂PO₄ potassium dihydrogen orthophosphate - Na₂HPO₄ disodium hydrogen phosphate - CaCl₂ Calcium Chloride - TCA Trichloroacetic Acid - PMSF Phenylmethylsulfonyl Fluoride     

SPECIFIC INHIBITION OF CELL PROLIFERATION IN THE MOUSE INTESTINE BY AN AQUEOUS EXTRACT OF RABBIT SMALL INTESTINE

An aqueous extract was prepared from the mucosa of rabbit small intestine by homogenization and centrifugation at 105,000 g. After precipitation with ammonium sulfate, the 0–50 fraction (F₁) and the supernatant (F₂) were collected, dialysed against a phosphate buffer and tested on rats in vitro and mice in vivo. The F₁ fraction was found to inhibit thymidine incorporation into rat intestinal DNA in vitro, but this effect was not found to be tissue specific (liver, kidney). Two hours after a single injection of F₁ (10 mg protein content), the uptake of tritiated thymidine was decreased in jejunal and colonic DNA in mice. This effect was maximal between 2 and 4 hr and totally reversible after 7 hr; this effect was found in neither the kidney nor the testis. A slowing of cellular migration was also noticed in the jejunum and the colon. Conversely, the F₂ fraction did not inhibit the synthesis of jejunal and colonic DNA either in vitro or in vivo. Our results suggest that the F₁ fraction of the aqueous extract of rabbit small intestine contains one or more substances which may act either on intestinal DNA synthesis or on the G₁–S transition of the cellular cycle in the mouse intestine. This reversible and specific intestinal action appears to inhibit cell proliferation and presents several of the characteristics defining a chalone.     

The bronchoalveolar lymphocyte: Studies on the life history and lymphocyte traffic from blood to the lung

Although recent work has shed some light on the identity and function of lymphocytes that reside in the bronchoalveolar air space (lung lymphocytes), little is known about the origin and life history of these cells. To determine the proportion of recently divided lung lymphocytes, DA-strain rats were labeled in vivo for 3 days with tritiated thymidine ([³H]dTR). Autoradiographs of lavaged lung and peritoneal cells indicated that a large fraction (44–77%) of lung lymphocytes was labeled and that these values were comparable to the proportion of labeled lymphocytes in peritoneal exudates (61–74%). To determine if some newly divided lung lymphocytes might come via the blood, additional experiments were performed in which rats were labeled ([³H]dTR) in vivo for 7 days. Lymphocytes were then obtained in labeled rats by thoracic duct drainage and were adoptively transferred (by vein) into syngeneic recipients. The percentage recovery of labeled lymphocytes in lung aspirates of recipient rats was determined from cell counts and autoradiographs. These results demonstrate that blood may be a source of recently divided lymphocytes but they do not indicate the relative contribution blood makes toward these cells in the lung.     

Lipopolysaccharide of Escherichia coli, polyamines, and acetic acid stimulate cell proliferation in intestinal epithelial cells

Summary Our aim was to examine whether lipopolysaccharide of Escherichia coli, polyamines of dietetic and/or bacterial origin, and products of the bacterial metabolism influence cell proliferation in epithelial cells from the colon and small intestine. Lipopolysaccharide of Escherichia coli 0111:B4 was incubated with cultures from human colonic mucosa. The mitoses were arrested with Vincristine and the total number of metaphases per crypt was counted. In addition, lipopolysaccharide was incubated with a human colonic epithelial cell line from adenocarcinoma (LS-123 cells) and with a nontransformed small intestinal cell line from germ-free rats (IEC-6 cells) for 24 h. In the last 4 h, the cells were labeled with tritiated thymidine. The cells were incubated with putrescine, cadaverine, and spermidine at 10⁻¹¹–10⁻³ M and with acetic acid (10⁻⁵–10⁻¹ M), acetaldehyde (10⁻¹⁰–10⁻⁴ M) and ammonium chloride (1–20 mM). Lipopolysaccharide of Escherichia coli increased the number of arrested metaphases in human colonic crypts and DNA synthesis in L-123 and IEC-6 cells (P<0.001). All polyamines increased DNA synthesis in the colonic and small intestinal cell lines, the effects being more marked for putrescine (P<0.001). The higher concentrations of acetic acid increased DNA synthesis in both epithelial cell lines (P<0.001). Acetaldehyde slightly decreased DNA synthesis in LS-123 cells at cytotoxic concentrations. Ammonium chloride did not significantly affect DNA synthesis. The final concentration of nonionized ammonia was less than 3%. It is concluded that lipopolysaccharides of Escherichia coli and intraluminal factors derived from microorganisms increase cell proliferation in human colonic crypts and intestinal epithelial cell lines.     

Epithelial cell proliferation and migration in the developing rat gastric mucosa

To investigate cell proliferation in developing gastric antrum and fundus, proliferating gastric epithelial cells were labeled in fetal rats by intravenously injecting mothers with [³H]thymidine. In addition 14-day postnatal (dPN) rats were given [³H]thymidine intraperitoneally. Tissue was removed 1.5 hr later, and autoradiographs were prepared to identify proliferating cells. Total epithelial labeling indices (L.I.) reached a peak at 20 days gestation (dG), coincident with the appearance of pit/glands, then declined again by 22 dG (gestation end). At 18 dG, labeled cells were distributed throughout all levels of the stratified epithelia. Between 20 and 22 dG, as pit/gland development proceeded, labeled cells became concentrated in the gland bases and were only rarely seen on the surface (L.I. of surface cells <1% at 22 dG). By 14 dPN, proliferating cells were entirely absent from the epithelial surface. Approximately 15% of the endocrine cells were labeled at 22 dG, compared to only 2% at 14 dPN; zymogen cells were labeled (～6%) at 14 dPN; parietal cells did not label at any age studied. In addition, cell migration to the epithelial surface was studied in rats labeled at 22 dG, 14 dPN, or 28 dPN, and killed 1–20 days later. Migration times were slightly shorter in the 28 dPN group and were longer in fundus than antrum in all groups.     

Mechanism of inhibition of proton: Dipeptide co-transport during chronic enteritis in the mammalian small intestine

Amino acids, a critical energy source for the intestinal epithelial cells, are more efficiently assimilated in the normal intestine via peptide co-transporters such as proton:dipeptide co-transport (such as PepT1). Active uptake of a non-hydrolyzable dipeptide (glycosarcosine) was used as a substrate and PepT1 was found to be present in normal villus, but not crypt cells. The mRNA for this transporter was also found in villus, but not crypt cells from the normal rabbit intestine. PepT1 was significantly reduced in villus cells also diminished in villus cell brush border membrane vesicles both from the chronically inflamed intestine. Kinetic studies demonstrated that the mechanism of inhibition of PepT1 during chronic enteritis was secondary to a decrease in the affinity of the co-transporter for the dipeptide without an alteration in the maximal rate of uptake (V_max). Northern blot studies also demonstrated unaltered steady state mRNA levels of this transporter in the chronically inflamed intestine. Proton dipeptide transport is found in normal intestinal villus cells and is inhibited during chronic intestinal inflammation. The mechanism of inhibition is secondary to altered affinity of the co-transporter for the dipeptide.     

The Jagged-2/Notch-1/Hes-1 Pathway Is Involved in Intestinal Epithelium Regeneration after Intestinal Ischemia-Reperfusion Injury

Background

Notch signaling plays a critical role in the maintenance of intestinal crypt epithelial cell proliferation. The aim of this study was to investigate the role of Notch signaling in the proliferation and regeneration of intestinal epithelium after intestinal ischemia reperfusion (I/R) injury.

Methods

Male Sprague-Dawley rats were subjected to sham operation or I/R by occlusion of the superior mesenteric artery (SMA) for 20 min. Intestinal tissue samples were collected at 0, 1, 2, 4, and 6 h after reperfusion. Proliferation of the intestinal epithelium was evaluated by immunohistochemical staining of proliferating nuclear antigen (PCNA). The mRNA and protein expression levels of Notch signaling components were examined using Real-time PCR and Western blot analyses. Immunofluorescence was also performed to detect the expression and location of Jagged-2, cleaved Notch-1, and Hes-1 in the intestine. Finally, the γ-secretase inhibitor DAPT and the siRNA for Jagged-2 and Hes-1 were applied to investigate the functional role of Notch signaling in the proliferation of intestinal epithelial cells in an in vitro IEC-6 culture system.

Results

I/R injury caused increased intestinal crypt epithelial cell proliferation and increased mRNA and protein expression of Jagged-2, Notch-1, and Hes-1. The immunofluorescence results further confirmed increased protein expression of Jagged-2, cleaved Notch-1, and Hes-1 in the intestinal crypts. The inhibition of Notch signaling with DAPT and the suppression of Jagged-2 and Hes-1 expression using siRNA both significantly inhibited the proliferation of IEC-6 cells.

Conclusion

The Jagged-2/Notch-1/Hes-1 signaling pathway is involved in intestinal epithelium regeneration early after I/R injury by increasing crypt epithelial cell proliferation.     

骨髓间充质干细胞动员内源性干细胞修复放射性肠黏膜损伤的实验研究

目的探索骨髓间充质干细胞（MSCs）对放射性肠炎（RE）肠黏膜修复的途径。 方法体外分离、培养大鼠骨髓MSCs。将RE模型的大鼠采用随机数字表法分为治疗组[经尾静脉注射干细胞悬液1 mL（细胞浓度为1×10⁶个/mL）]和对照组（注射等量生理盐水,每日1次,连续3 d）,每组10只。每日观察两组大鼠的活动量、进食和进水量、体质量变化等。1周后处死大鼠获取小肠标本,HE染色观察肠黏膜的修复情况,电镜观察上皮细胞的超微结构,免疫组化染色观察小肠隐窝Bmi-1阳性干细胞增殖情况,采用Image-Pro Plus 6.0软件对Bmi-1阳性细胞数量进行分析。组间差异的比较采用t检验。 结果成功分离得到大鼠骨髓MSCs,流式细胞仪鉴定：CD29、CD90、CD34、CD45阳性细胞比例分别为98.6﹪、99.6﹪、0.56﹪、0.89﹪。与对照组相比,治疗组大鼠移植1周后,体质量增加（190.30 g ± 13.23 g比235.00 g±14.30 g）;大鼠小肠黏膜上皮得到修复,绒毛高度增高（627.50 μm ± 40.55 μm比984.33 μm ± 61.80 μm）;上皮细胞超微结构较完整、清晰,隐窝Bmi-1阳性干细胞增殖数量增多[（60.67±9.63）个/mm²比（87.33 ±5.47）个/mm²],差异具有统计学意义（P < 0.05）。 结论骨髓MSCs能促进RE模型的大鼠肠黏膜的修复,这一作用可能是通过促进小肠隐窝干细胞的增殖发挥。