비기능항진증이 골수에 미치는 영향에 관한 자기방사법적 연구

Abstract

[한글]

비장의 기능항진이 골수에 미치는 영향을 추구하고자, egg-albumin 반복투여방법에 의

거 가토에게서 실험적 비기능항진증을 유발시켜 혈액학적 관찰을 하고, 골수를 채취하여

in vitro로 배양하였으며, 동시에 액성인자의 존재 가능성을 보기 위하여 정상 및 비기능

항진 혈청을 혼합 배양하고, H**3-thymidine(H**3-Th.)을 이용한 자기방사법을 시행하여

다음과 같은 결과를 얻었다.

Egg-albumin의 투여로 현저한 비종대와 함께 빈혈과 혈소판감소가 초래되었으나, 망상

구와 백혈구는 별 변화가 없었다. H**3 -Th.표지율은 비기능항진골수배양군에서 정상 및

비적출 골수배양군에 비하여 현저히 낮았고, 정상골수배양군에 비기능항진혈청을 혼합배

양하면 감소하였다. 반대로 비기능항진골수배양군에 정상혈청을 혼합 배양하면 증가하였

다. H**3-Th.표지치는 정상 및 비기능항진 골수배양군이 서로 유사하였으며, 비적출골수

배양군에서는 훨씬 낮았다. 그러나 정상과 비기능항진 혈청을 혼합배양한 군 사이에는 별

차가 없었다. 추산된 DNA 합성시간은 각 골수배양군간에 별 차이가 없었으나 비적출골수

배양군에서는 단축되었다. 세포생산시간은 비기능항진골수배양군에서 현저히 연장되었고,

비적출골수배양군에서는 약간 단축되었으며, 정상골수배양군에 비기능항진혈청을 첨가하

면 역시 현저하게 연장되었다. 그리고 정상 및 비기능항진 혈청을 혼합한 비기능항진골수

배양군에서도 현저하게 연장되었으나, 양군간의 차는 별로 없었다.

따라서 이상과 같은 결과를 종합 검토하면 비기능항진증에서의 혈구감소의 성인은 골수

세포의 증식과 성숙이 장애되기 때문이라 추리되며, 이러한 억제작용은 아마도 종대된 비

장에서 유리되어 혈액내에 존재하고 있는 어떤 액성인자에 기인하는 것이라 생각된다. 그

러나 이 기전만이 혈구감소의 유일한 성인이라고 단정할 수는 없다 하겠다.

[영문]The prob1em in hyperactivity of the spleen, since the day of Gretzel (1866) and

Banti (1882), has been plagued by uncertainty of meaning, doubt of etiology and

capriciousness of therapeutic results. In 1907, Chauffard introduced a term

"hypersplenism" and Dameshek in 1941 divided it into primary and secondary:

selective and total types.

The term encompasses various disorders in which blood cytopenia is associated

with splenomegaly, thus the cardinal features of the hypersplenism are: (1)

splenomegaly, which may be of unknown etiology (primary) , or secondary to some

known disease processes for splenic growth: (2) a reduction of one or more cellular

elements of the blood leading to anemia, leukopenia, thrombocytopenia, or any

combination of these : (3) hypercellular marrow with hyperplasia of the respective

marrow precursors of the deficient cell type : and (4) temporary or permanent

correction of the blood cytopenia (5) by splenectomy, even if it does not alternate

the basic disease process.

Since the Banti's hypotheses that the spleen destroys large number of blood

cells, or that certain biochemical processes taking place in the spleen results in

the development of certain metabolites which enter the circulation and have

secondary effects on hematopoietic organs, the controversy over the pathogenesis of

hypersplenism continues until the present time, and the hematology literature still

carried extensive debate and laboratory investigation supporting one or the other

of these two possibilities. Thus Dameshek (1941, 1955) concluded that the spleen

releases humours which suppress the production or release of new blood cells from

the marrow into circulation, whereas Dean (1949) argued that large spleens have an

enhanced capacity for sequestering blood cells, thereby lowering the peripheral

blood counts of these elements, and Rambach & Alt (1962) proposed an autoimmune

concept viewing hypersplenism as manifestations of autoimmune diseases.

Many attempts to induce hypersplenism in experimental animals have been

conducted, such as ligation of splenic and gastric coronary veins. administration

of silica gel, polyvinyl alcohol, gum acasia, pectin, hydroxylamine, antirabbit

marrow serum, tubercle bacilli, tryptophan blue, zymosan, methylcellulose, human

gamma-globulin and egg-albumin, but the autoradiographic technique in the study of

experimental hypersplenism has been utilized seldomly.

Therefore, the purpose of present study is to observe the effect of egg-albumin

induced hypersplenism on the bone marrow proliferation with autoradiographic

technique using H**3 -thy-midine in rabbits.

Materials and Methods

Hematologic observation: Rabbits weighing about 2kg, regardless of sex, were

divided into three groups : normal control, hyperaplenic and asplenic. The

hypersplenic group received daily injections of 1% egg-albumin solution 1ml and

asplenic group treated same way from two weeks after splenectomy. Hematologic

examinations including hemoglobin, hematocrit, reticulocyte, leukocyte and platelet

count were done with approximate;y two weeks interval.

Autoradiographic observations : On 97th day after injections of egg-albumin, the

bone marrow was obtained by femoral puncture under ether anesthesia, aspirated into

a heparinized syringe and immediately diluted with the same volume of culture

medium 199-Ⅸ (Earle's base). One ml of the bone marrow suspension was transferred

into pre-siliconized culture tubes. According to the following bone marrow culture

groups- (1) normal bone marrow (NM), (2) hypersplenic bone marrow(HSM), (3)

asplenic bone marrow(ASM) : (4) normal bone marrow with normal serum(NM+7NS) , (5)

normal bone marrow with hypersplenic strum(NM+HSS) : (6) hypersplenic bone marrow

with normal serum (HSM+NS), and (7) hypersplenic bone marrow with hypersplenic

serum (HSM+HSS) group, 0.5 ml of normal or hypersplenic serum obtained by cardiac

puncture at the time of bone marrow aspiration was added respectively, and then

H**3-thymidine (0.062 mg, 1 m1/1 mCi) was added to a final concentration of 2.5μCi

per milliliter. The procedure was carried out under sterile conditions and earth

bone marrow culture group was consisted of 3 culture tubes. Incubating the tuhea at

37℃., samples taken from each tube at 1, 3, 5, 10, and 24 hours smearer on

gelatin-coated slides, fixed in Carney's solution, and after several washing in

distilled water, autoradiographic preparations were made with Kodak Fine-Grain

Autoradiographic Stripping Plate AR-10 according to the technique described by Pelc

(1956). After an exposure of 7 days, the firms were developed and stained through

stripping film with Giemsa.

One thousand nucleated cells observed and grains over the cell nucleus was

estimated as scores as table 1 in the text. Fer each autoradiograph, (1)

differential count of cells, (2) per cent labeled cells, and (3) scores per labeled

cell were counted, then (4) DNA synthetic time and (5) generation time for each

cell type in each group were deduced.

Results and Conclusions

The results obtained are summarized as follows:

1. Repeated intravenous injecitons, of egg-albumin to rabbits induced

splenomegaly, anemia and thrombocytopenia without significant changes of

reticulocyte and leukocyte count, whereas only mild thrombocytopenia was noted in

pre-splenectomized rabbits.

2. There was no significant difference of differential cell count in each group.

3. Per cent labeled cells was markedly reduced in HSM group and somewhat

increased in ASM group. It was also reduced in NM+HSS group while there was no

change in NM+NS group. Though it was decreased in HSM+NS group and HSM+HSS group,

it twas higher in the former.

4. Scores per labeled cell baa similar in NM and HSM group, but significantly low

in ASM group. There was no significant difference either between NM+NS and NM+HSS

group, or HSM+NS and HSM+HSS group.

5. DNA synthetic time showed no significant difference between the groups, but it

was shortened in ASM group.

6. Generation time was significantly prolonged in HSM group and slightly

shortened in ASM group. It was also markedly prolonged in NM+HSS group while there

was no change in NM+NS group. There was marked prolongation of it in both HSM+NS

and HSM+HSS group, but no significance between these two groups.

7. Such differences observed with autoradiographic technique were remarkable in

the erythroid elements but indistinct in the myeloid elements.

The results obtained from present study indicate that the cytopenias induced by

egg-albumin administration in rabbits are mostly due to defects in proliferation

and maturation of the bone marrow cells-especially erythroid series, and this

inhibitory effect nay be attributed to possible humoral factor(s) released from the

enlarged spleen and present in the serum. But it cannot be concluded whether this

inhibitory effect is the only mechanism far the cytopenia(s) in hypersplenism or

not.