الفهرس 
AIM OF THE WORKOnly 14 pages are availabe for public view
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Abstract
SUMMARY
Liver fibrosis, which is the main cause of many chronic liver diseases, is primarily characterized by an extensive deposition of ECM proteins (mainly type 1 collagen) in response to hepatic damage. The accumulation of collagen interferes with hepatic architecture and function which may subsequently progress into cirrhosis and liver failure. One commonly used hepatotoxin in the experimental studies of liver diseases is carbon tetrachloride (CCl4). It is a carcinogenic agent and induced genotoxicity in which its metabolites cause DNA strand break, chromosome aberrations (CAs) and sister chromatid exchanges.
Advanced fibrosis is generally considered to be irreversible condition even after removal of the injurious agent. Moreover, the development of fibrosis is characterized by a diminution of liver collagenases activity, implying that the capacity of the diseased liver to remodel the fibrotic matrix is critically reduced. Currently, the only effective clinical therapy for end-stage liver diseases is orthotropic liver transplantation. However, several defects, such as surgical complexity, immunological rejection, low survival rate and particularly the shortage of donors’ liver, preclude it from active use in clinics.
Use of stem cells (SCs) to cure liver diseases has been proved beneficial in most of the conditions. Scientific literature reveals the role of stem cells in treatment of various diseases like Liver Cirrhosis, End Stage liver Failure, genetic liver disease and also the Liver cancer. Stem cell therapy can be mediated by either embryonic, induced pluripotent or adult stem cells (ASCs). Ethical issues and concerns make reduce the use of embryonic stem cells (ESCs) as a source to cure liver diseases when compared to ASCs. Mesenchymal stem cells (MSCs) represent a promising tool for new clinical concepts in supporting cellular therapy. Bone marrow (BM) was the first source reported to contain MSCs. However, for clinical use, BM may be detrimental due to the highly invasive donation procedure and the decline in MSCs number and differentiation potential with increasing age. More recently, umbilical cord blood (UCB), attainable by a less invasive method, was introduced as an alternative source for MSCs.
The aim of the present study was to evaluate the therapeutic effect of BM-MSCs and UCB-hematopoietic CD34+ stem cells in treating hepatic fibrosis and diminishing the percentage of chromosomal abnormalities that caused by CCl4 administration. This study was performed at the Genetic Lap of Zoology Department of Women’s College, Ain Shams University and also in the Unit of Biochemistry and Molecular Biology of the Medical Biochemistry Department, Faculty of Medicine, Cairo University, Cairo, Egypt.
This work was divided as follows:
Isolation and characterization of rat BM-MSCs morphologically and by detection of CD29 gene.
Isolation and identification of UCB-CD34+ hematopoietic stem cells using a magnetic cell sorter.
In vitro labeling of isolated two types of SCs with florescent PKH26 dye.
Preparation of rat model of liver fibrosis by CCl4-induced injury
SCs administration.
qRT- PCR for type 1 Collagen and MMP-2 gene expression.
Biochemical analysis for liver functions.
Examination of liver histopathology and immunohistochemistry.
Cytogenetic study on BM cells of rats to estimate percentage of structural and numerical CAs.
Statistical analysis.
The present work was carried out on 60 male albino rats of 6 weeks old, weighing between 150 and 200 g. Liver fibrosis was induced by CCl4 injected by subcutaneous route at a dose of 0.2 mL/100 g body weight of 40 mL/L CCl4 dissolved in equal volume of castor oil. The injection was given twice a week for 6 weeks. After 6 weeks of CCl4 administration, stem cells (MSCs and CD34+) were given as a single dose of 107 cells per rat and left for one month. Rats were divided into the following groups:
1- Healthy control group: rats received 0.2 mL/100 g body weight of castor oil twice a week for 6 weeks.
2- Positive control group: rats received 0.2 mL/100 g body weight of CCl4 twice a week for 6 weeks.
3- CCl4/MSCs group: rats received CCl4 firstly for 6 weeks then infused with a dose of 107 MSCs per rat IP.
4- CCl4/CD34+ group: rats received CCl4 firstly for 6 weeks then infused with a dose of 107 CD34+ cells per rat IP.
At the end of experiment, venous blood was collected from the eye of all rat groups for biochemical analysis. All rats were sacrificed with Diethyl Ether, liver tissue samples were removed for molecular, histopathological and immunohistochemical studies. Also, BM samples were harvested for cytogenetic examination.
The results stated that, CCl4 administration induced many histological alterations in the liver such as hepatocyte damage, vacuolar degeneration, necrosis, inflammation, congested and dilated blood vessels, hemmorrhage and fibrosis. Fibrosis spreads to link the vascular structures that feed into and drain the hepatic sinusoid (portal tract and central vein). CCl4 administration also causes activation of hepatic stellate cells (HPSCs) which display a myofibroblast phenotype and histologically distinguished by a significant increase (P<0.05) in α-SMA value compared to healthy control group. In addition, there was also a significant decrease (P<0.05) in the Alb level (2.33 ± 0.59) compared to healthy control group (3.88 ± 0.50) and significant increase (P<0.05) in the level of serum ALT (17.02 ± 3.06) after CCl4 administration compared to healthy control group (12.86 ± 2.39).
CCl4 also resulted in a significant increase (P<0.05) in type 1 collagen gene expression (1.09 ± 0.27) compared to healthy control group (0.13 ± 0.03) and MMP-2 gene expression (0.94 ± 0.17) compared to healthy control group (0.24 ± 0.06). The results also revealed that, rats treated with CCl4 developed significant genetic damage (P<0.05) as evidenced by an increase in the frequency of total chromosomal abnormalities (70.88 ± 10.71) when compared to healthy control group (0.88 ± 1.13). The frequency of structural CAs was 122.5 % and the most frequent types were chromatid gap (25.2 %) and deletion (19.5 %). Also, the numerical CAs increased after induction of CCl4 (19.2 %) and the hypoploidy (12.5 %) was the most frequent type.
In the present work, it has been shown that treatment with rat BM-MSCs and human UCB-CD34+ cells decreased hepatic injury and improved liver tissue architecture of rats given CCl4 as assessed by reduction in the fibrotic matrix, necrosis, hepatocyte degeneration, congestion and dilation of blood vessels and hemorrhage and an increase in hepatic cells regeneration. Also, the value of α-SMA which is a marker of activated HPSCs was reduced after 4 weeks of SCs transplantation but still non-significant (P>0.05) when compared to CCl4 group. In addition, there was non-significant difference (P>0.05) between stem cell groups and healthy control group, and also there was no significant difference (P>0.05) between the two groups treated with the two types of SCs.
The results also showed a significant elevation (P<0.05) in serum Alb level after administration of MSCs (3.33 ± 0.49) and CD34+ stem cells (3.47 ± 0.59) compared to CCl4 group (2.33 ± 0.59). Additionally, there was no significant difference (P>0.05) in CD34+ stem cells group compared to healthy control group, and statistically there was no significant difference (P>0.05) between the two groups treated with the two types of SCs. The results also detected a significant decrease (P<0.05) in ALT level in the group that received CD34+ stem cells (10.2 ± 2.68) compared to CCl4 group (17.02 ± 3.06), meanwhile there was a slight decrease in ALT level in the group that received MSCs (15.73 ± 3.74) and still non-significant (P>0.05) when compared to CCl4 group. Also, there was non-significant difference (P>0.05) in MSCs and CD34+ treated groups when compared to healthy control group, while there was a significant decrease (P <0.05) in ALT level in the group that received CD34+ stem cells comparing with MSCs treated group.
Moreover, type 1 collagen gene expression was significantly decreased (P<0.05) in MSCs (0.50 ± 0.22) and CD34+ stem cells (0.51 ± 0.23) groups compared to CCl4 group (1.09 ± 0.27). Additionally, MMP-2 gene expression was significantly decreased (P<0.05) in the groups that treated with MSCs (0.40 ± 0.15) and CD34+stem cells (0.46 ± 0.22) compared to CCl4 group (0.94 ± 0.17), and there was no significant (P>0.05) difference between the two groups treated with the two types of SCs.
Also, treatment with CD34+ stem cells significantly decreased (P<0.05) the total CAs in the bone marrow cells to 21.63 ± 3.42 compared to CCl4 group (70.88 ± 10.71), and also there was non-significant difference (P>0.05) when compared to healthy control group. Meanwhile, there was a slight decrease in the total CAs in CCl4/MSCs group (25.50 ± 4.50) and still non-significant (P>0.05) comparing with CCl4 group. Administration of MSCs after CCl4 injury reduced frequency of structural CAs to 41.7 % and numerical CAs to 9.2 %. Also, treatment with CD34+ stem cells after CCl4 injury reduced frequency of structural CAs to 38 % and numerical CAs to 5.2 %. Statistically, there was no significant (P>0.05) difference between the two groups treated with the two types of SCs.
from the present study it can be concluded that, MSCs and CD34+ multipotent cells have ameliorative ability against injury in the liver of rats treated with CCl4 and subsequently promote the regeneration process and the production of liver cells and lead to improvement in liver functions. SCs transplantation were shown to improve the injurious effect of CCl4 where they could be prevent the formation of scar lesions in the liver and also ameliorate the chromosomal damage in BM cells of rats intoxicated with CCl4. The present study also revealed that, there was better improvement in the therapeutic potency of human CD34+ SCs in the restoration of liver function and amelioration of chromosomal damage. However, the improvement was less than expected due to xenogeneic transplantation