Experimental establishment of in situ and subcutaneous transplantation models of human liver malignant lymphoma in nude mice

Summary

The establishment of in situ and subcutaneous transplantation models of human hepatic malignant lymphoma in nude mice can (1) explore the pathogenesis of hepatic malignant lymphoma, (2) provide a tool for experimental therapies, and (3) provide tumor animal models for experimental studies on the etiology and pathogenesis of hepatic malignant lymphoma.

Operation method

In situ and subcutaneous graft models

Principle

Human liver malignant lymphoma was implanted into the liver parenchyma and interscapular subcutis of nude mice with fresh tissue blocks from the operation to observe the tumorigenicity rate of in situ transplantation and subcutaneous transplantation, invasion and metastasis of the transplanted tumors, and to perform morphological (light microscopy, electron microscopy, immunohistochemistry), serological [alpha-fetoprotein (AFP), Hepatitis B Virus Surface Antigen (H BsAg), lactic acid dehydrogenase (LDH)], karyotypic and flow cytometric analyses. Karyotype and flow cytometric analysis.

Materials and Instruments

BALB C-nu nu nude mice Fresh specimen of liver malignant lymphoma
RPMI-1640 culture medium Tincture of iodine Alcohol Sodium pentobarbital CD3 antibody CD7 antibody CD19 antibody CD20 antibody CD45RO antibody CD79a antibody
Silk thread Optical microscope Casing needle Scalpel Gauze

Move

I. Preparation of experimental animals

BALB/C-nu/nu nude mice, aged 4~6 weeks, weighing 17~20 g, both male and female, were reared under specific-pathogen free (SPF) conditions in the nude mouse room of our hospital.
Sources of specimens

1. The fresh specimen of hepatic malignant lymphoma came from a 53-year-old male patient, whose preoperative laboratory tests were normal for liver function, AFP, CEA, and bone marrow image, except for elevated LDH (1,200 U/L) and positive HBsAg. 2.

2. The operation confirmed multiple nodular lymphoma of the liver, with multiple nodules in the right lobe of the liver and multiple nodules in the left lobe fused into a 5.3 cm x 3.5 cm x 3.0 cm mass, which was grayish-white and fish-like in color, with yellowish-white in the middle of the nodule and red irregular congested bands in the periphery.

3. There was no other tissue or organ invasion or involvement of distant lymph nodes during the operation, and the pathological diagnosis of the tumor nodule in the left lobe of the liver was non-Hodgkin's B-cell-derived malignant lymphoma of the liver and chronic hepatitis.
Specimen processing

Aseptically cut human liver malignant lymphoma biopsy, put into RPMI-1640 culture solution, remove the non-tumor tissue, and cut it into 1 mm x 1 mm x 1 mm tissue pieces for transplantation.
Subcutaneous transplantation
The skin on the back of nude mice was sterilized with 2.5% tincture of iodine and 75% alcohol, and two 1 mm x 1 mm x 1 mm lumps of tumor tissue were aspirated with a sterile cannula needle and transplanted into the subcutaneous tissue on the back of the neck.

V. In situ transplantation
1. Nude mice were anesthetized with sodium pentobarbital (30 mg/kg) intravenously, a transverse incision was made in the left upper abdomen to expose the liver, the peritoneum of the left lobe of the liver was incised, and two 1 mm x 1 mm x 1 mm tumor blocks were implanted into the hepatic parenchyma. The hepatic peritoneum was closed by 10-0 nondestructive threads, the peritoneum was closed by 7-0 threads, and the skin was sewn up by 5-0 threads.

2. After the operation, we continued to feed and observe the growth of the tumor day by day.

Transmission

1. When the tumor-bearing nude mice are in a state of near-death, take one of the tumor-bearing nude mice and execute it by cervical dislocation, systematically dissect it and take out the transplanted tumor. 2.

2. One part of the transplanted tumor tissue will be transmitted to the other mice by the method of primary transplantation, with 5~10 nude mice each time; the other part of the tumor tissue will be frozen in liquid nitrogen for spare use, and will be used for the detection of relevant indexes. 3.

3. The other tumor-bearing nude mice were kept until near death or natural death to observe the growth, invasion and metastasis of the tumors.

VII. Observation Items
1. Anatomical and histological examination

(1) All executed and naturally dead nude mice with tumor were subjected to detailed anatomical examination, and the growth of intrahepatic tumors and subcutaneous graft tumors were recorded.

(2) Lymph nodes were sampled from the para-iliac arteries, inter-iliac arteries, mediastinum, mesentery, pylorus, cardia and hepatic hilar lymph nodes, etc. Organs such as lungs, livers, spleens, kidneys, brains and other organs were routinely sampled, and all the tissues were fixed with 10% neutral formaldehyde, paraffin sections were cut, stained with hematoxylin, eosin and light microscopy was carried out.

2. Immunohistochemical staining

Immunohistochemical staining was performed on all liver tumor sections of nude mice with CD3, CD7, CD19, CD20, CD45RO, CD79a antibodies using LSAB method.The reagents used were purchased from DaKo Company.
3. Electron microscopic observation

The transplanted tumors were double-fixed with 2.5% glutaraldehyde and 1% osmium acid, embedded in Epon-812, ultrathin sectioned, stained with uranium and lead, and observed by transmission electron microscope with PHILIPS-CM10.
4. Laboratory tests

AFP immunoassay; Hepatitis B markers: HBsAg, HBeAg, HBsAb, HBeAb, HBcAgELISA; LDH-L method.
5. Flow cytometry analysis

Flow cytometer (FACS-420) was used to measure the DNA content of normal hepatocytes, tumor-derived specimens and transplanted tumor cells.
6. Chromosome examination

The cells were cultured for a short period of time without mitotic stimulation, and the harvested cells were prepared routinely, stained with GTG method and observed under the microscope.
7. Histologic classification of malignant lymphoma.

The new WHO classification of 2001 should be adopted.
8. Statistical treatment
The significance of differences was determined by t-test, and the data were expressed as x±s, which was done on SPSS 10. 0 statistical software.

Caveat

It is because the liver microenvironment of nude mice is similar to that of human liverThe similarity of the implanted tumors to human liver provides an ideal animal model for basic and clinical studies of primary malignant lymphoma of the liver, as the implanted tumors appear to be similar to the dissemination process of human liver primary malignant lymphoma.

Common Problems

MLL accounts for 0.016% of non-Hodgkin's lymphoma and is a rare malignant tumor. The etiology and pathogenesis of MLL have not yet been elucidated, and the clinical onset of MLL is insidious, with a lack of specificity in symptoms, signs, and auxiliary examinations, and it is confusing with primary hepatocellular carcinoma or liver metastases, which makes it difficult to diagnose, with a low rate of preoperative diagnosis, a high rate of misdiagnosis, and poor prognosis. Therefore, both basic and clinical studies of MLL require the establishment of an ideal in situ transplantation model of human liver primary malignant lymphoma in nude mice for research. The establishment of an in situ transplantation model of human liver primary malignant lymphoma in nude mice has not been similarly reported both at home and abroad after an international on-line search. In the present study, we successfully established a human liver malignant lymphoma in situ transplantation model HLBL-0102 in nude mice for the first time by adopting a histologically intact human liver malignant lymphoma fresh tissue block transplanted in situ in nude mice, which was completely confined to the liver. this model is valuable for the study of the biological characteristics, histogenesis, experimental treatment, tumor immunity, and molecular biology of MLL. The successful establishment of this model creates favorable conditions for the systematic development of MLL diagnosis, surgery, radiotherapy, chemotherapy, targeted gene therapy and drug screening and other prospective experimental treatments, which are not easy to obtain MLL tissue specimens for research. At the same time, it also provides fresh tumor tissue specimens and animal models of the same human source, which are stable and easily reproducible for the study of basic theories of MLL.

The ability to maintain the original biological characteristics of human primary lymphoma in nude mice after in situ transplantation of human liver primary lymphoma is the key to the establishment of the model. Our model HLBL-0102 has been transmitted to 37 generations and grown in nude mice for 3 years. Not only the histopathology, immunohistochemical phenotype, ultrastructure, DNA ploidy level and chromosomal characteristics of the transplanted tumor were identical to those of the source MLL cells, but also the growth rate, fluid content, and the tumor growth rate of the transplanted tumor were identical to those of the source MLL cells. Moreover, the growth rate of the transplanted tumors and the survival rate of liquid nitrogen cryopreservation were 100%. The transplanted tumors did not regress spontaneously, and always proliferated according to the pattern of latency, slow growth and fast growth, and the survival time of the hosts was close to the death of the hosts, which was always in the malignant state, which indicated that the biological characteristics of the model were stable.

It is worth proposing that transplanted tumors demonstrate their malignant behavior in the host in a manner similar to that of MLL patients. We found that the HLBL-0102 transplant tumor was completely confined to the liver, which was enlarged in multiple nodules, and the section was scattered with numerous white tumor nodules with fused parenchymal masses between the nodules, and remnants of hepatic tissue were visible between the nodules, and in severe cases, the hepatic tissue was replaced by tumor nodules. There was no invasion of other tissues and organs or involvement of distant lymph nodes, which fully met the criteria for the diagnosis of MLL. In the experiment, it was also found that HLBL-0102 completely simulated the in vivo manifestations of human MLL: the peripheral blood AFP negativity, HBsAg and HBeAg positivity, and lactate dehydrogenase elevation found in MLL patients were all seen in nude mice transplanted with tumors in situ. Moreover, LDH levels increased significantly with the prolongation of the growth period and tumor enlargement in the nude mice. The results of HBsAg test suggested that human liver malignant lymphoma retained its original positive characteristics after transplantation in nude mice. Moreover, the authors observed that the tumor cells diffusely infiltrated the liver, destroying the confluent area, the bile ducts and the hepatic lobules, and a large number of mature lymphocytes infiltrated the confluent area around the tumor foci. This experiment also confirmed the suggestion by Maher et al. that the histogenesis of MLL originates from lymphocytes in the hepatic confluent zone and that continuous stimulation by chronic hepatitis virus plays an important role in the histogenesis of MLL.Elevated LDH is a sensitive indicator for the diagnosis of MLL and for determining the prognosis of MLL. These results indicate that HLBL-0102 completely reproduces the clinicopathological features and clinical biological behaviors of MLL.

In summary, the reasons for the high tumor growth rate and extensive intrahepatic dissemination in the nude mice in situ transplantation model of human primary malignant lymphoma in the liver are: (1) the high invasive and malignant potential of the tumor cells; and (2) the microenvironmental factors of the transplantation site, such as the special structure of the liver, the rich lymphatic and blood circulation, and the biochemical and immunological characteristics of the transplantation site. The similarity between the liver microenvironment of nude mice and that of human liver is precisely the reason why the implanted tumors appeared similar to the dissemination process of primary malignant lymphoma in human liver, which provides an ideal animal model platform for the basic and clinical research of primary malignant lymphoma in the liver.

Reference Zhang N, Tuo Shuai, Liu Qiu Zhen et al. Establishment and biological characterization of in situ and subcutaneous transplantation models of human liver malignant lymphoma in nude mice. Digestive Surgery , 2006 (5)

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