الفهرس 
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Abstract
Egypt has the highest prevalence of HCV in the world (predominantly genotype 4), which has been attributed to previous public health eradication schemes for schistosomiasis. Even higher HCV infection rates, up to 60%, have been reported in older individuals, in rural areas such as the Nile delta, and in lower social classes. In Egypt, HCC was reported to account for about 4.7% of chronic liver disease (CLD) patients. Between 1993 and 2002, there was an almost two-fold increase in HCC amongst chronic liver patients in Egypt.
Hepatocellular carcinoma (HCC) is one of the most frequent and lethal human cancers, and its incidence is rising. HCC has an overall dismal prognosis and only when diagnosed early, surgery and ablative therapies may offer a cure. In most cases, however, HCC is diagnosed at an advanced stage, the multi tyrosine kinase inhibitors (sorafenib or regorafenib) and most recently the immune checkpoint inhibitor nivolumabor remain the only available treatment options. To develop more effective therapies and to identify factors that determine patient survival, a better mechanistic understanding of HCC is urgently needed.
Different Liver diseases can be treated using stem cells like liver cirrhosis, genetic liver diseases and liver necrosis. In response to liver injury or loss of liver mass, proliferation of mature liver cells is the first-line defense to restore liver homeostasis.
In the present study mesenchymal stem cells were isolated umbilical from cord and identification was done by flow cytometer, while cultures of human hepatoma cell line (HuH-7) was performed on those from passage 14 from VACSRA. The cells were cultured in two kind of media. Conditioned medium is the medium in which umbilical cord mesenchymal stem cells were cultured then diluted by DMEM media (1:1).Co-culture medium is that collected after mesenchymal stem cells and HuH7 cells (1:1) together were incubated for 3days.
Morphological characterization of HuH7 after treatment with conditioned media showed that culturing in conditioned media for 48hrs led to appearance of dead cells and cells were changed in shape. After 72 hrs the number of dead cells increased and cells were dissociated while after 96hrs a larger number of dead cells were observed. After treatment with co-culture media for 48hrs appear a number of dead cells and cells were dissociated. After 72 hrs the number of dead cells increased and cells were more dissociated while after 96hrs number of dead cells increased greatly.
The above results indicate that both the conditioned media and co-culture media had great effect on cell viability and survival of HuH7 cells and that was time dependent. The Ultrastructure study of HuH7 cultured in the normal media, conditioned media and co-culture media showed that there are various changes between the cases. In conditioned media for 48hrs the cells showed complete absence of filopodia with either loss of the plasma membrane or appearance of few blebs. Most of the cells suffered from high vacuolation, loss of RER and ribosomes, decrease in number of intact mitochondria which sometimes are cristolysed. In Conditioned media for 72 hrs cells showed increased lipidosis where most of the cells appeared filled with large amount of unsaturated lipid droplets and globules. Advanced lytic necrosis was also noticed .where large number of cells lost their plasma membrane and their nuclei were karyolysed. In Conditioned media for 96 hrs all examined fields showed that all cells lost their filopodia, their plasma membrane and endoplasmic reticulum while fat droplets were still found. Lytic necrosis and apoptosis were more frequently observed. Most of the nuclei suffered from karyolysis. Like the other treatment cases with conditional media no glycogen was detected in any of the cells. Mitochondria were swollen and greatly cristolysed. Numerous amounts of phagosomes were also noticed. Most of the cells appeared as moth-eaten areas. Peroxisomes were frequently noticed but not in all cells examined. In co culture media for 48 hrs all cells suffered from absence of plasma membrane, high increase of lipid globules and leakage of all organelles outside the cells. Mitochondria were swollen and lost their membrane integrity. Apoptosis was more frequently observed with more cell fragments. Complete absence of glycogen was noticed in all cells examined, while lipid globules increased greatly in size and occupy most of the cytoplasm. In co culture media for 72hrs there were cell fragments and apoptotic bodies. Rarely remnants of damaged cells could be seen where the cytoplasmic mass noticed was filled with irregular vacuoles indicating lytic necrosis and no plasma membrane could be distinguished.
By using the MTT assay, it was found that there are highly significant differences in the viability between HuH7 cultured in normal media, HuH7 cultured in the conditioned media (p<0.001) and co-culture media (p<0.001).
In the present study we measured some biochemical parameters such as Alfa fetoprotein as tumor marker, albumin, GOT, GPT, Alkaline phosphatase and ƔGT in HuH7cultured in normal media and HuH7 cultured in conditioned and co-culture media at different time intervals (0, 48hrs, 72hrs and 96hrs).
The marker alfa fetoprotein showed that there is highly significant difference between the cases at the different time intervals and that was obvious by comparing the alfa fetoprotein at the four time intervals in HuH7 in normal media and those cultured in conditioned and co-culture media.
The study of albumin showed that there was non-significant difference between the two groups (conditional and co-culture groups) (p=0.054).
Liver enzyme GOT showed highly significant difference between the four groups (0-time, 48hrs, 72 hrs and 96 hrs) in case of groups cultured in conditioned media (p<0.001) and those cultured in co-culture media (p<0.001). The data showed that the GOT activity is inversely proportional with increase of time since the values decrease with time and at 96hrs the cells show the lowest value.
There was no significant differences between the four groups (0-time, 48 hrs, 72 hrs and 96 hrs) in GPT activity in case of groups cultured in conditioned media(p<0.674) and those cultured in co-culture media (p<0.674).
The study of Alkaline phosphatase showed non-significant difference between the four groups (0-time, 48 hrs, 72 hrs and 96 hrs) in case of HuH7 cells cultured in conditional media (p=0.294) nor in case of culturing in co- culture media (p=0.127).
The measure of ƔGT showed non-significant difference between the two groups (conditional and co-culture groups) (p=0.844).
Gene expression for Survivin, PCNA, β-Catenin, Telomerase and VEGF in HuH7 was analysed in this study.
The gene expression of Survivin, PCNA, Beta Catenin, Telomerase and VEGF was performed by using real time PCR at several time intervals (48-72-96hrs). In conditioned media and co-culture media, hepatoma cell line (HuH7) demonstrated a significant down regulation in Survivin (p<0.001), PCNA (p<0.001), VEGF (p<0.001) and Beta Catenin (p=0.022) but not significant in Telomerase (p=0.617).
CONCLUSION
T
he present work showed the potential clinical applications of conditional media and co-culture media of human UCMSCs on a model of HCC. Ultrastructural investigations affirms that future studies should focus on determining the role played by exosomes and their potential diagnostic and therapeutic benefits in cancer treatments. Biochemical results and gene expression analysis support the conception that conditioned and co-culture media greatly suppressed and structurally damaged cancer cells.